The chromosomal constitution of wine strains of Saccharomyces cerevisiae

A general procedure is described for determining the chromosomal constitution of industrial strains of Saccharomyces cerevisiae based on analysis of segregation frequencies for input markers among random spore progeny of industrial-laboratory strain hybrids. The multiply auxotrophic haploid testers used carried a dominant erythromycin-resistance marker, allowing hybrids to be selected in mass matings with spores produced by the wild-type industrial strains. Analysis of a number of independent crosses between the haploid testers and an unselected population of spores of each wine strain distinguished between disomic, trisomic and tetrasomic chromosomal complements in the parents. Possible explanations for a significant class of aberrant segregation frequencies are discussed. Results of the analysis indicate that UCD Enology 522 (Montrachet) is diploid and possibly trisomic for chromosome VII; 522X is diploid; UCD Enology 505 (California Champagne) is disomic for chromosome XVI, trisomic for chromosomes I, II, III, VI, VIII, IX, X, XII, XV, tetrasomic for chromosomes IV, XI, XIII, XIV and either trisomic or tetrasomic for chromosomes V and VII; and that UCD Enology 595 (Pasteur Champagne) is disomic for chromosomes I, II, III, IX, XVI, trisomic for chromosomes IV, VI, X, XII, XIV, XV, tetrasomic for chromosomes V, VIII, XI, XIII and either disomic or tetrasomic for chromosome VII.     

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.     

Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.     

Genetic homology between Saccharomyces cerevisiae and its sibling species S. paradoxus and S. bayanus: electrophoretic karyotypes.

Chromosomal DNAs of many monosporic strains of the biological species Saccharomyces cerevisiae, S. paradoxus and S. bayanus were analysed using contour-clamped homogeneous electric field electrophoresis. Southern blot hybridization with eight cloned S. cerevisiae genes (ADC1, CUP1, GAL4, LEU2, rDNA, SUC2, TRP1 and URA3) assigned to different chromosomes was used to study homology and chromosomal location of the genes in the three sibling species. A comparative study of Ty1, Ty2 and telomere-associated Y' sequences having multiple chromosomal location was also done. Chromosome length polymorphism was found in cultured strains of S. cerevisiae. Wild S. cerevisiae and S. paradoxus strains yielded chromosome banding patterns very similar to each other. The karyotype pattern of S. bayanus was readily distinguishable from that of S. cerevisiae and S. paradoxus. Southern blot analysis revealed a low degree of homology between the S. cerevisiae genes studied and the corresponding S. paradoxus and S. bayanus genes. The number of chromosomes appears to be 16 in all three species.     

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.     

Assignment of most genes encoding major peroxisomal polypeptides to chromosomal band V of the asporogenic yeast Candida tropicalis

The peroxisomes of the asporogenic yeast Candida tropicalis contain about 20 major polypeptides (PXPs). We have isolated a number of genes encoding them; 11 POX genes encoded independent PXPs and three POY genes were likely to encode three other PXPs. To locate these genes on the chromosomes, chromosomes of C. tropicalis were separated by pulsed-field gel electrophoresis. Eight chromosomal bands were observed over the range of 1.0 Mbp (band 1) to 2.8 Mbp (band VIII); the genome size was estimated to be about 20 Mbp. Southern blot analysis showed that ten genes were on band V, three genes were on band IV, and the other gene was on band VI. Three genes gave hybridization signals of nearly equal intensity on two different chromosomal bands: POX6A and POX8B, on bands V and VII; and POX8A, on bands IV and VI. Ribosomal RNA genes also hybridized to two bands, VI and VII. Most genes assigned to only one band hybridized to two restriction fragments produced by either NotI or SfiI endonuclease. The results suggested that C. tropicalis was diploid and that restriction sites were conserved little between homologues. The three POX genes that were found on two chromosomal bands hybridized to not more than two restriction fragments, implying that the allelic genes were present on different chromosomal bands.     

Creating a Saccharomyces cerevisiae haploid strain having 21 chromosomes

Chromosome engineering techniques that can manipulate a large segment of chromosomal DNA are useful not only for studying the organization of eukaryotic genomes but also for the improvement of industrially important strains. Toward the development of techniques that can efficiently manipulate a large segment of chromosome, we have previously reported a one-step chromosome splitting technique in a haploid Saccharomyces cerevisiae cell, with which we could successfully split yeast chromosome 11, XIII, or XI into two halves to create a haploid strain having 17 chromosomes. We have now constructed chromosome splitting vectors bearing ADE2, HIS3, LEU2, or TRP1 marker, and by using these vectors, we could successively split yeast chromosomes to create a novel yeast haploid strain having up to 21 chromosomes. The specific growth rates of yeast strains carrying more than 16 chromosomes up to 21 did not differ significantly, suggesting that yeast cells can harbor more chromosomes than they do in their natural state, that is, 16 chromosomes, without serious effects on their growth.     

Chromosome translocation induced by the insertion of the URA blaster into the major repeat sequence (MRS) in Candida albicans

Electrophoretic karyotype studies have shown that clinical isolates of Candida albicans have extensive chromosome length polymorphisms. Chromosome translocation is one of the causes of karyotypic variation. Chromosome translocation events have been shown to occur very frequently at or near the major repeat sequence (MRS) on chromosomes. The MRS consists of the repeated sequences RB2, RPS and HOK, and the repeated sequences are considered to be the template for recombination. To investigate which element of the MRS is important for chromosome translocation, we constructed three cassettes, each containing a URA blaster and sequences homologous to one of the repeats, for insertion into the MRS region on the chromosomes. The ura3 strain STN22u2, which shows a stable, standard karyotype, was transformed with each construct. Insertion events with each cassette occurred at almost all chromosomes. Insertion into the RB2 repeat, but not into the RPS repeat, was accompanied by chromosome translocation in some transformants: chromosome translocations between chromosomes R and 7 and chromosomes 1 and 7 were found, as well as deletions of 7A and 7C from chromosome 7. We conclude that the insertion at the RB2 region may initiate chromosome translocation in C. albicans.     

Disruption and mapping of IDI1, the gene for isopentenyl diphosphate isomerase in Saccharomyces cerevisiae.

Isopentenyl diphosphate isomerase catalyses an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for isomerase, IDI1, was recently isolated and characterized (Anderson et al. J. Biol. Chem. 1989a, 264, 19169-19175). Wild-type IDI1 was disrupted with a LEU2 marker, and the resulting DNA was used to transform a yeast leucine auxotroph. Southern blots of EcoRI fragments of chromosomal DNA from the diploid strain showed the expected fragments for intact and disrupted IDI1. Dissection and analysis of tetrads demonstrated that IDI1 is an essential single-copy gene. A CHEF gel and clone grid filter analysis, followed by chromosomal mapping indicated that the gene is located on chromosome XVI approximately 55 kb centromere proximal to PEP4.     

Genetic mapping of the α-galactosidase MEL gene family on right and left telomeres of Saccharomyces cerevisiae

The α-galactosidase MEL2–MEL10 genes have been genetically mapped to right and left telomere regions of the following chromosomes of Saccharomyces cerevisiae: MEL2 at VII L, MEL3 at XVI L, MEL4 at XI L, MEL5 at IV L, MEL6 at XIII R, MEL7 at VI R, MEL8 at XV R, MEL9 at X R and MEL10 at XII R. A set of tester strains with URA3 inserted into individual telomeres and no MEL genes was used for mapping.     

Polymorphism of Saccharomyces cerevisiae aquaporins

Aquaporin water channels facilitate the transmembrane diffusion of water and higher organisms possess a large number of isoforms. The genome of the yeast Saccharomyces cerevisiae contains two highly similar aquaporin genes, AQY1 and AQY2. AQY1 has been shown to encode a functional water channel but only in certain laboratory strains. Here we show that the AQY2 gene is interrupted by an 11 bp deletion in 23 of the 27 laboratory strains tested, with the exception of strains from the sigma 1278b background, which also exhibit a functional Aqy1p. However, although the AQY2 gene from sigma 1278b is highly homologous to functional aquaporins, we did not observe Aqy2p-mediated water transport in Xenopus oocytes. A survey of 52 yeast strains revealed that all industrial and wild yeasts carry the allele encoding a functional Aqy1p, while none of these strains appear to have a functional Aqy2p. We conclude that natural and industrial conditions provide selective pressure to maintain AQY1 but apparently not AQY2.     

Mapping of the trifunctional fatty acid synthetase gene FAS2 on chromosome XVI of Saccharomyces cerevisiae

The trifunctional FAS2 gene encoding subunit alpha of the Saccharomyces cerevisiae fatty acid synthetase complex was mapped on the left arm of chromosome XVI 24 centimorgans proximal to GAL4 and 39 centimorgans distal to PEP4 relative to the centromere. Mapping was achieved by three independent methods: meiotic co-segregation of FAS2 and ARO7 in recombination-deficient spo11-mutants: tetrad analysis of crosses between FAS2, GAL4 and PEP4; and Southern hybridization of purified FAS2 DNA with individual yeast chromosomes separated by pulsed-field gel electrophoresis.     

Duplication of the beta-galactosidase gene in some Lactobacillus plantarum strains.

Curing of a plasmid that encoded a beta-galactosidase gene (beta-gal) from the Lactobacillus plantarum strain of dairy origin LL441 was not accompanied by complete loss of the lactose utilization phenotype. DNA-DNA hybridization, using an internal fragment of the beta-gal gene as a probe, revealed a second determinant located on the chromosome of the cured derivatives. The chromosomal copy was present in all of a series of beta-Gal+ L. plantarum and Lactobacillus pentosus strains from different origins. In addition, four other L. plantarum strains harboured plasmid encoded beta-gal genes as well. Since both sequences cross-hybridized and present a similar genetic organization, it is postulated that the plasmid copy was generated through gene duplication and, probably, selected by growth of the strains in lactose rich environments.     

Brewing characteristics of haploid strains isolated from sake yeast Kyokai No. 7

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai No. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small‐scale sake‐brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups, indicating fermentation ability and sake flavour. Copyright © 2008 John Wiley & Sons, Ltd.     

PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae

Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra- or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome.     

The alpha-aminoadipate pathway for lysine biosynthesis is widely distributed among Thermus strains

We previously reported that lysine is synthesized through the alpha-aminoadipate pathway in Thermus thermophilus HB27 (T. Kosuge and T. Hoshino, FEMS Microbiol. Lett., 169, 361-367, 1998), which was the first report demonstrating the synthesis of lysine through the alpha-aminoadipate pathway in Bacteria. LYS20 and LYS4, which respectively encode homocitrate synthase and homoaconitate hydratase have already been identified as the lysine biosynthetic genes in T. thermophilus HB27. In the present work, we examined eight other Thermus strains for the existence of genes belonging to the alpha-aminoadipate pathway. BamHI- or BglII-digested total DNAs from the eight strains were analyzed by Southern hybridization using LYS20 or LYS4 as a DNA probe. DNA fragments that hybridized with one or both of the genes were detected in seven of the Thermus strains but not in T. ruber. The sizes of the fragments that hybridized with the LYS20 and LYS4 probes were the same among T. thermophilus HB27, T. thermophilus HB8, "T. caldophilus" GK24, and four "T. flavus" strains. For example, a similar 4.3-kb fragment was detected in each of the above seven strains. In this fragment, four open reading frames were found downstream of the LYS4 gene in T. thermophilus HB27. Gene disruption experiments revealed that three open reading frames are involved in lysine biosynthesis in T. thermophilus HB27. These results strongly suggest that the lysine biosynthetic gene cluster for the alpha-aminoadipate pathway is widely distributed in the genus Thermus.     

APPLICATION OF PULSED FIELD CHROMOSOME ELECTROPHORESIS IN THE STUDY OF CHROMOSOME XIII AND THE ELECTROPHORETIC KARYOTYPE OF INDUSTRIAL STRAINS OF SACCHAROMYCES YEASTS

Pulsed field chromosome electrophoresis is a powerful new technique in yeast genetics which permits the resolution of intact yeast chromosomes in an agarose gel matrix. We utilized contour-clamped homogeneous electric field electrophoresis (CHEF) to survey representative strains of Saccharomyces yeasts from the brewing, baking, distilling, sake and wine industries for their electrophoretic karyotypes. All of the strains tested were found to have a unique chromosomal profile, indicating the potential of this technology for “fingerprinting” prototrophic strains of Saccharomyces yeasts. By employing an ILV2 gene probe specific for chromosome XIII, we determined that all of the industrial strains of Saccharomyces yeasts possessed a chromosome XIII which migrated in an identical fashion to chromosome XIII from a reference haploid strain of Saccharomyces cerevisiae. While one lager yeast strain, Saccharomyces carlsbergensis M244, was found to contain two alleles of ILV2 when digested genomic DNA was probed with ILV2, the presence of a novel independently migrating chromosome XIII could not be detected. A homeologous chromosome XIII in this yeast will therefore have to be determined by genetic analysis. Pulsed field chromosome electrophoresis is concluded to be a technology with immediate application to Quality Control and Research and Development programs in industries using Saccharomyces yeasts.     

Efficient removal of sulfide following integration of multiple copies of the sulfide-quinone oxidoreductase gene (sqr) into the Escherichia coli chromosome

For the oxidation and removal of hydrogen sulfide, which causes an offensive odor from the contents of animal intestines, recombinant strains of Escherichia coli were constructed. The sulfide-quinone oxidoreductase gene (sqr) from Rhodobacter capsulatus was integrated in low copy numbers into the chromosome of Escherichia coli W3110. Multiple copies of sqr on plasmids were also delivered into the cytoplasm of the same strain. The sqr genes were homologously transducted onto the chromosomal lacZ region and their existence there was verified by Southern blot analysis. Sulfide oxidation in a chemical medium effectively increased for the recombinant strains which carried 2 approximately 3 copies of sqr under the control of the lac or tac promoter in the chromosome, and also for strains which carried 10 copies of sqr under the control of the lac or tac promoter on plasmids. In both types of recombinant, the tac promoter was more effective for SQR expression than the lac promoter. Construction of a recombinant with 3 copies of sqr under the control of the tac promoter in the chromosome was unsuccessful. In recombinants with SQR activity lower than 700 nmol/mg cell protein/min, oxygen consumption increased proportionally to SQR activity. An elevation in SQR activity in this range resulted in an increase in oxygen consumption and a decrease in sulfide concentration. When the recombinant cells were cultured until the 160th generation, WL2, WL3 and WT2, which carried 2, 3 and 2 copies of sqr in the chromosome, respectively, retained SQR activity similar to that of the first generation. For WL300 and WT20 which carried multi-copies of sqr in plasmids SQR activity was undetectable. The recombinant with 2 copies of sqr in the chromosome regulated by the tac promoter was most suitable for sulfide oxidation and growth of the cells.     

Cloning and analysis of the AWA1 gene of a nonfoaming mutant of a sake yeast

Almost all sake yeasts form a thick foam layer on sake mash during fermentation. To reduce the amount of foam, nonfoaming mutants were bred from foam-forming sake yeasts. To elucidate the mechanism of this foam formation, we have cloned a gene from a foam-forming sake yeast that confers foam-forming ability to a nonfoaming mutant. This gene, named AWA1, encodes a glycosylphosphatidylinositol (GPI) anchor protein that is localized to the cell wall and is required for cell surface hydrophobicity. In this paper, we describe the genomic analysis of the AWA1 gene in a nonfoaming mutant strain K701 derived from a foam-forming sake yeast strain K7. K701-AWA1 was cloned in a cosmid and its sequence was compared with that of K7-AWA1. Although the 5' half of K701-AWA1 was identical to that of K7-AWA1, the 3' half of K701-AWA1 was different from that of K7-AWA1, resulting in a loss of the C-terminal hydrophobic sequence of Awa1p. Since this sequence is considered to be required for the anchoring of Awa1p to the cell wall, K7-Awa1p could not confer both cell surface hydrophobicity and foam-forming ability to strain K701 cells. Since the change found in K701-AWA1 was not a point mutation but a larger scale event, we analyzed chromosome rearrangement by pulsed-field gel electrophoresis Southern blot analyses. The results suggest that the left subtelomeric region of chromosome IX in strain K7 was translocated to the AWA1 gene in chromosome XV by a nonreciprocal recombination.