DNA RESEARCH 18, 423–434, (2011)
Advance Access Publication on 6 September 2011

doi:10.1093/dnares/dsr029

Whole-Genome Sequencing of Sake Yeast Saccharomyces cerevisiae
Kyokai no. 7

National Research Institute of Brewing, Higashi-hiroshima, Hiroshima 739-0046, Japan1; National Institute ofTechnology and Evaluation, Shibuya-ku, Tokyo 151-0066, Japan2; Department of Applied Molecular Bioscience,
Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi 755-8611, Japan3; Kagoshima Prefectural
Institute of Industrial Technology, Kagoshima 899-5105, Japan4; Department of Biotechnology, Graduate School ofEngineering, Osaka University, Suita, Osaka 565-0871, Japan5; Nihonsakari Co., Ltd, Nishinomiya, Hyogo
662-8521, Japan6; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto
611-0011, Japan7; Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute ofTechnology, Yokohama, Kanagawa 226-8501, Japan8; Department of Biotechnology, University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan9; Food Research Center, Aichi Industrial Technology Institute, Nagoya, Aichi
451-0083, Japan10; Central Laboratories for Frontier Technology, Kirin Holdings Co., Ltd, Yokohama, Kanagawa
236-0004, Japan11; Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University,
Fukuoka 812-8581, Japan12; Gunma Industrial Technology Center, Maebashi, Gunma 379-2147, Japan13;
General Research Laboratory, Kiku-Masamune Sake Brewing Co., Ltd, Kobe, Hyogo 658-0026, Japan14; R&D
Planning Division, Products Development Center, Suntory Holdings Ltd, Kawasaki, Kanagawa 211-0067, Japan15;
Department of Fermentation, Faculty of Applied Bio-Science, Tokyo University of Agriculture, Setagaya-ku, Tokyo
156-8502, Japan16; Research Institute, Gekkeikan Sake Co., Ltd, Kyoto 612-8385, Japan17; Biotechnology andFood Research Institute, Fukuoka Industrial Technology Center, Kurume, Fukuoka 839-0861, Japan18; ResearchLaboratories of Brewing Technology, Asahi Breweries, Ltd, Moriya, Ibaraki 302-0106, Japan19; Frontier
Laboratories of Value Creation, Sapporo Breweries Ltd, Yaizu, Shizuoka 425-0013, Japan20; School of Pharmacy,
Hyogo University of Health Sciences, Kobe, Hyogo 650-8530, Japan21; Food Technology Research Center, Hiroshima
Prefectural Technology Research Institute, Hiroshima 732-0816, Japan22; General Research Laboratory, Ozeki
Corporation, Nishinomiya, Hyogo 663-8227, Japan23; Research and Development Department, Hakutsuru Sake
Brewing Co., Ltd, Kobe, Hyogo 658-0041, Japan24 and Brewing Society of Japan, Kita-ku, Tokyo 114-0023,
Japan25*To whom correspondence should be addressed. Tel. þ81 82-420-8016. Fax. þ81 82-420-0809.
E-mail: simoi@nrib.go.jp
Edited by Katsumi Isono(Received 25 January 2011; accepted 26 July 2011)

Abstract
The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possesscharacteristics distinct from others including the laboratory strain S288C and are well suited for sakebrewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sakeyeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the
S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of# The Author 2011. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.
org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work
is properly cited.

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TAKESHI Akao1, ISAO Yashiro2, AKIRA Hosoyama2, HIROSHI Kitagaki1, HIROSHI Horikawa2, DAISUKE Watanabe1,
RINJI Akada3, YOSHINORI Ando4, SATOSHI Harashima5, TOYOHISA Inoue6, YOSHIHARU Inoue7, SUSUMU Kajiwara8,
KATSUHIKO Kitamoto9, NORIYUKI Kitamoto10, OSAMU Kobayashi11, SATORU Kuhara12, TAKASHI Masubuchi13,
HARUHIKO Mizoguchi14, YOSHIHIRO Nakao15, ATSUMI Nakazato16, MASAHIRO Namise17, TAKAHIRO Oba18,
TOMOO Ogata19, AKINORI Ohta9, MASAHIDE Sato20, SEIJI Shibasaki21, YOSHIFUMI Takatsume7,
SHOTA Tanimoto22, HIROKAZU Tsuboi23, AKIRA Nishimura24, KOJI Yoda9, TAKEAKI Ishikawa25,
KAZUHIRO Iwashita1, NOBUYUKI Fujita2, and HITOSHI Shimoi1, *424

Genome Sequencing of Sake Yeast[Vol. 18,

heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven
distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeatedlosses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both
K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the twostrains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty
elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNAmediated recombination of Ty elements. The present study advances our understanding of the functionaland evolutionary genomics of the sake yeast.
Key words: Saccharomyces cerevisiae; sake yeast; genome sequence; diploid; loss of heterozygosity
Introduction

Sake is a traditional Japanese alcoholic beveragethat is fermented from steamed rice by the concerted
action of two types of microorganisms, filamentous
fungi and yeast. In the production of sake, enzymes
secreted by the fungus Aspergillus oryzae, which isgrown on steamed rice, convert rice starch into
glucose. Yeast cells in the sake mash then produce
ethanol, higher alcohols and their esters, organic
acids and amino acids, which are important components that contribute to sake aroma and taste.
Hence, the choice of a yeast strain is one of themost critical factors in determining the resulting
aroma and taste characteristics of sake products.
Yeast strains that were originally isolated in sake breweries have been identified as Saccharomyces cerevisiae
and are now commercially distributed as sake yeast.1
Phylogenetic studies conducted using DNA markers
have indicated that sake yeast strains are closely
related, forming a sake strain cluster that belongs to alineage distinct from other industrial and laboratory
strains in the phylogenetic tree of S. cerevisiae.2 – 5Comprehensive genome-wide studies of diverse
S. cerevisiae strains have also indicated the existence ofa unique sake cluster that is distinct from wine and laboratory strains.6,7 Consistent with their unique phylogenetic position, sake yeast strains possess characteristic
traits that differ from other S. cerevisiae strains andare ideal for sake brewing, including ability for highethanol productivity (reaching 20%)1,8,9 efficient
growth and fermentation at low temperatures (below
158C).1 In addition, nearly all sake yeast strains generatea foam on the mash surface during the brewing process,which results from yeast cells fermenting sugars
into CO2 bubbles10 and possess biotin biosynthetic
ability.11
Several sake yeast-specific genes that are responsible
for the desirable features of sake yeast and affect thebrewing process have been identified. For example,
AWA1, which encodes a cell-surface hydrophobic
protein with a GPI anchor, was identified from a sakeyeast genome and is responsible for foam formationin sake mash.10 Many S. cerevisiae strains are biotin

auxotrophs due to the lack of certain biotin biosynthetic pathway genes. BIO6, a homolog of a bacterial
biotin biosynthetic pathway gene, was identified insake yeast strains and is essential in them for the production of biotin.11 In addition to these studies,
several genes involved in yeast’s quantitative phenotypes, including fermentability and aroma production,
have been suggested.12,13 However, because thegenetic basis for the superiority of sake yeast in sakebrewing is largely unknown, genome-wide genetic
approaches are required to understand these complex
traits. Since the first complete genome sequencing ofstrain S288C in 1996,14 the genomes of several otherS. cerevisiae strains have been sequenced.15 – 18 Strains
such as K11, Y9 and Y12 were also subjected togenome sequencing and the sake cluster was consequently proposed. However, in each case, total lengthof sequence reads reached ,0.9-fold of the haploid
genome, not enough to get whole picture of the
genome.6 In addition, these sequenced strains are not
the typical industrial sake yeast strains. Accordingly,
yeast strains used in sake brewing have not been subjected to whole-genome analyses, despite their importance in industry and yeast phylogenetic systematics.
In the present study, we performed the wholegenome sequencing of the sake yeast strain K7(kyokai is the Japanese word for ‘society’) as the firststep for subsequent functional, phylogenetic and evolutionary genomic studies. K7 has been one of themost extensively used industrial sake yeast strains
over the past several decades and has also been
employed in numerous genetic and biochemical
studies as a model sake yeast and a parent strain forbreeding.8,10 – 13,19 – 24 Here, we report the overallchromosome structure and remarkable features of
the K7 genome.
2.

Materials and methods

2.1. Strain
Saccharomyces cerevisiae K7 was distributed toJapanese sake breweries by the Brewing Society ofJapan in 2004 and used as a DNA donor.

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2.3. Sequence comparison
An overall comparison of the K7 chromosomeswith S288C (NC_001133– NC_001148), EC1118(FN393058– FN393060, FN393062 – FN393087,
FN394216 and FN394217) and YJM789 (AAFW
2000000) strain chromosomes was performed
using MUMmer 3.0 software.29 Similarity-based
searches of individual genes were performed using
BLAST30 and BLAST2.31 Phylogenetic analyses werecarried out using CLUSTALW 1.83.32
2.4. Extraction of heterozygous positions
To determine the positions of heterozygosity in theK7 genome, nucleotide positions containing one ormore bases different from the consensus base with
the Phred quality values .20 were automaticallyextracted as candidates for heterozygous positions.
The identified positions were then manually validated
on the electropherogram to validate the sequence.
Non-supercontig contigs, which failed to be assembled
into supercontigs, were not included in the analysis
because their chromosomal positions were unknown.

direct comparison of S288C ORFs with the K7
genome supercontigs. When direct comparison was
difficult, ORFs were predicted using the software programs CRITICA,33 Glimmer2,34 GlimmerHMM35 andSIM4.36 Finally, all K7 ORFs were manually validated
by expert annotators. When one or more incomplete
ORFs, such as those truncated by a sequence gap andlacking a start or a stop codon, were mapped to asingle S288C ORF, each incomplete K7 ORF was annotated as a single ORF. Functional annotation was based
primarily on the Saccharomyces Genome Database(SGD; http://www.yeastgenome.org/), secondarily on
the Saccharomyces species database (yeast comparative
genomics: http://www.broadinstitute.org/annotation/
fungi/comp_yeasts/) and also on COG/KOG(http://www.ncbi.nlm.nih.gov/COG/) and DDBJ/EMBL/
GENBANK non-redundant databases. Orthology with
the S288C ORF was evaluated using the BLASTP
similarity and calculated as the percent of matchedamino acid residues versus the total covered region
between a K7 ORF and the best-hit S288C ORF(Supplementary Table S4) as truncated by a sequencegap. Similarity was calculated by the number of matching residues in only the corresponding regions of the
S288C ORF. Dubious ORFs, ORFs in Ty elements and
ORFs in telomeric regions were excluded as possible
protein-coding genes and were not annotated.
Prediction and annotation of RNA genes, Ty elements
including solo long terminal repeats (LTRs) and telomericelements were manually performed based on the resultsof BLASTN searches of the K7 genome with the S288C
sequences of these genes and elements as queries.
All annotated ORFs and genetic elements were given
individual numbers (Supplementary Table S4).
Nomenclature of the K7 genes was based on the following rules: (i) each protein-encoding or RNA gene wasnamed according to the orthologous S288C geneusing the format ‘K7_’ plus the S288C standard genename (with .80% similarity) and the systematicname (with .50% similarity) given in SGD; (ii) K7
identification numbers or K7 original gene names,such as AWA1, were given to genes that were non-orthologous or of low similarity to S288C genes (with 50%
similarity); (iii) each name of a gene truncated by asequence gap or segmented by point mutations wasfollowed by a lower case ‘a’, ‘b’ or ‘c’, such as ‘XXX1a’and ‘XXX1b’, to show its correspondence to a partial
region of the ortholog; and (iv) Ty elements and LTRs
were independently termed according to the identicalnomenclature used for S288C.
3.

2.5. Gene prediction and annotation
For predicting protein-encoding genes, ORFs larger
than 90 bp were comprehensively included as candidates. ORF prediction was then carried out based on a

Results and discussion

3.1. Sequencing and assembly of the K7 genome
Genome sequencing of the diploid sake yeast strain
K7 was performed by a whole-genome shotgun

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2.2. Sequencing, assembly and validation
The nucleotide sequence of the K7 genome was determined using the whole-genome shotgun sequencing
approach. Genomic DNA was isolated from cells according to the method described by Hereford et al.25 Plasmid
libraries with average insert sizes of 1.6 and 5.0 kb wereconstructed in pUC118 (Takara Bio Inc.), while a fosmid
library with an average insert size of 35 kb was constructed in pCC1FOS (Epicentre Biotechnologies), as
described previously.26 Raw sequence reads corresponding to a 9.1-fold coverage of the haploid genome(42 842, 88 830 and 15 227 reads from libraries with
1.6, 5.0 and 35 kb inserts, respectively) were firstobtained by sequencing from both ends of the inserts
on an ABI 3730xl DNA Analyzer (Applied Biosystems).
Sequence reads were trimmed at a threshold quality
value (Phred) of 20 and assembled using the Phrap
assembler.27,28 We obtained a total of 712 contigs
that were then put in order based on paired-end information from the constructed fosmid library to obtainsupercontigs. The overall assembly was then validated
and refined using Optical Mapping (OpGen, Inc.). A
number of short contigs were incorporated into supercontigs with the assistance of optical maps and transposon-mediated random sequencing from fosmid clones(2112 reads). Following this analysis, the final contig
number was reduced to 706.

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Genome Sequencing of Sake Yeast

chromosomes due to base substitution, in-del, Ty insertion or other such events (Supplementary Fig. S1C and
D). Several non-supercontig contigs contained ORFlike sequences that were not found in the supercontigs.
For example, the nucleotide sequence of MATa wasfound in a non-supercontig contig due to its considerable heterozygosity, while the supercontig corresponding to the K7 chromosome III only contained the
MATalpha sequence, even though K7 possesses bothmating-type loci, MATalpha and MATa. Contigs including
the sequence corresponding to VTH1/VTH2, which areparalogous and nearly indistinguishable from eachother, were not assembled in the supercontigs, presumably due to adjacent repetitive sequences. Similarly, thesequence for ARR3 was also present only in the nonsupercontig contigs (data not shown). The non-supercontig contigs were excluded from subsequent analyses
such as comparison with related strains, gene prediction
and the survey of heterozygosity and Ty elements.
To date, the genome sequences of several S. cerevisiae
strains have been reported; therefore, we compared theK7 genome with available genomes.14,15,17 Pairwise
nucleotide polymorphisms among four strains (K7,S288C, YJM789 and EC1118) were analyzed bysequence alignment using MUMmer 3.0 software.29
The number of substitutions and small indels
between K7 and the three other strains ranged from67 900 (5.6/kb) to 78 000 (6.5/kb) and 19 300(1.6/kb) to 23 500 (2.0/kb), respectively, while
those among the remaining three non-K7 strains
ranged from 46 100 (3.8/kb) to 56 700 (4.7/kb)
and 14 800 (1.2/kb) to 16 700 (1.4/kb), respectively (Supplementary Fig. S2). These results indicate

Figure 1. Dot-plot alignments of homologous chromosomes of S. cerevisiae strains K7 and S288C. The nucleotide sequence of each K7
supercontig was compared with that of the homologous S288C chromosome (indicated above each alignment) using MUMmer 3.0.

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method that yielded 1.49  105 sequence reads withan estimated 9.24-fold redundancy of the haploid
genome. Following de novo sequence assembly, 17supercontigs were generated. Since homologous
chromosome pairs of K7 were almost indistinguishable from each other during the assembly process,nearly all the reads from each homologous chromosome pair were assembled together into a single
supercontig. Consequently, the resulting consensus
chromosomal sequences represented a diploid
genome, although they seemed to be that of a
haploid genome. The total length of the supercontig
corresponded to 98.1% of the estimated K7 genome
size. The sequencing results and the assembled supercontigs are summarized in Supplementary Tables S1and S2. Comparison with the S288C genome revealed
that the 17 supercontigs corresponded to the set of
S288C chromosomes and mitochondrial DNA(Fig. 1; Supplementary Table S2). No DNA sequence
for the yeast 2-mg plasmid was detected from any
reads, which was consistent with a previous study.37
In the assembly process, only 200 of 706 contigs wereused for generating the supercontigs because theremaining 506 contigs failed to align with any of thesupercontigs. The total length of these non-supercontig
contigs was 604 kb. Of the 506 non-supercontig
contigs, 360 (71.1%) were singletons and 395(78.1%) contained ,1000 bases (Supplementary Fig.
S1A and B). The majority of these contigs seem to be
excluded from the supercontigs due to the repetitionof telomeric and Ty-related type sequences. The
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T. Akao et al.

427that the phylogenetic position of K7 is relatively distantfrom that of S288C, YJM789 and EC1118, as expectedfrom previous studies.2,3,5 – 7,17

3.3. Uneven distribution of heterozygosity
The sequence obtained for the K7 genome represents the consensus haploid sequence derived
from the two homologous chromosomes, although
K7 is a diploid. Accordingly, we surveyed the positionsof heterozygosity by carefully examining the sequencereads. A total of 1347 heterozygous sites between thehomologous chromosomes were detected, and theirpositions were subsequently mapped (Fig. 2).
Interestingly, the windows containing multiple heterozygous sites were unevenly distributed as clusters
in several chromosomal regions.
Simple evolutionary accumulation of point
mutations is insufficient to explain such uneven distribution of heterozygosity.41 It is more likely that a heterozygous diploid was generated by the out-crossingof two different haploid strains and subsequent loss

Figure 2. Genome-wide distribution of heterozygosity between
homologous chromosomes of K7. Heterozygous sites wereidentified by manually checking sequence reads assembled
within the supercontigs. The frequency of the extracted
heterozygosity in homologous K7 chromosomes was plottedby each 10 kb window of the chromosomal coordinates. x-axis,
chromosomal corrdinates; y-axis, heterozygosity counts; arrowheads, position of centromeres.

of heterozygosity (LOH), resulting in the observed
pattern. Alternatively, a complex history of out- and/or back-crossings of the ancestral strain in naturalenvironments could have also caused this pattern.
However, repeated backcrossing is unlikely to haveoccurred in nature since the sporulation efficiency ofsake yeast, including K7, is markedly low. Thus, we
speculate that sequential LOH events have resulted
in the uneven distribution of heterozygosity in K7.
Conversely, it is reasonable to presume that isolated
heterozygosities were introduced by point mutations
independent of LOH events. Genome-wide LOH was

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3.2. Inversions in chromosomes V and XIV
We compared chromosomal structures between K7
and S288C using MUMmer 3.0 software,29 as shownin Fig. 1. Although the overall genome structure of
K7 closely resembled that of S288C, we identified
two types of chromosomal rearrangements. One
type involved several complicated subtelomeric
rearrangements that are also observed or suggested
by previous genome-wide studies of various S. cerevisiae
strains, indicating that such rearrangements were notinfrequent events.14,17,18,38 – 40 The other type ofrearrangement was characterized by two large
internal inverted regions. We confirmed that these
inversions were homozygous by PCR analysis (datanot shown). One inversion of the 100-kb region
on the right arm of chromosome V had not previously
been described. Both boundary regions of this inversion on the K7 chromosome V were flanked by two
Ty2 elements (K7_YERCTy2-3 and K7_YERWTy2-4)that were inverted in relation to each other andwere absent in the corresponding region of S288C.
Therefore, these Ty2 elements have been proposed
to mediate this reciprocal inversion (Supplementary
Fig. S3).15 Since Ty insertions differ by strain or
lineage, this inversion would be unique to K7 or
related lineages (Supplementary Fig. S3). The second
identified inversion was a 30-kb region on the left
arm of chromosome XIV. This inversion was also
observed in other strains.15,17 Inverted homologous
regions located close to the breaking points on
chromosome
XIV(YNL018C – YNL019C
andYNL033W – YNL034W) may have mediated this reciprocal inversion.

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Genome Sequencing of Sake Yeast

3.4. Gene predictions
Following the sequencing and assembly of the K7
genome, we predicted and annotated 5815 ORFs on
16 nuclear chromosomes and mitochondrial DNA(Supplementary Table S4). We observed many incomplete ORFs interrupted by a sequence gap between
contigs: 39 ORFs had a truncated terminal at one
end and 124 ORFs (62 pairs) had internal gaps.
When compared with the S288C genome, frame
shifts caused by small indels and single-nucleotidechanges at the start or the stop codons resulted inmany ORF polymorphisms, including terminal disagreement, such as extension or truncation (132
genes), segmentation of a single S288C ORF into multiple K7 ORFs (89 genes corresponding to 43 orthologs in S288C) and the fusion of ORFs (13 genes
corresponding to 26 orthologs in S288C;
Supplementary Table S4). The influence of these polymorphisms on each respective gene function is
unclear and remains to be elucidated, although forseveral cases, such as the K7 ortholog of MSN4, polymorphism appears to influence the characteristic features of K7.43
The average BLAST similarity between K7 ORFs andthe most similar S288C ORFs was greater than 95%(95.7 and 95.8% at the nucleotide and amino acidlevels, respectively). More than 90% of the K7 ORFs
displayed a similarity exceeding 97%, with the corresponding S288C ortholog at the amino acid level(Fig. 3). Genes for tRNAs and other non-coding RNA
were also annotated (Supplementary Table S3),
which revealed that they corresponded to an almost
complete set of S288C RNA genes. However, K7 lost
three tRNA genes and one copy of two RUF5 ncRNA
genes (data not shown). Their absence would not be

Figure 3. Identities of K7 ORFs with the top-hit S288C ORF. Each K7
ORF was compared with the corresponding orthologous S288C
ORF using BLASTN (nucleotide level) and BLASTP (amino acidlevel). The ORF proportion based on the observed identities
was then plotted.

expected to have an effect on the cellular functionsof K7 because tRNA genes corresponding to a specificcodon are highly redundant and one copy of RUF5
was still present in the K7 genome.
3.5.

Differentially present genes between K7 and
S288C
The comparison of ORFs between K7 and S288C
genomes disclosed 97 differentially present genes:
48 ORFs unique to K7 and 49 ORFs unique to
S288C (Supplementary Tables S5 and S6). In thisanalysis, we excluded differences in subtelomeric multicopy gene families such as PAU, COS, DAN, SNO and
MAL. Many of the unique genes were located at subtelomeric plastic regions of K7 and S288C, indicatingthat they may have been acquired or lost by chromosomal rearrangement events. Most differentiallypresent genes located in internal chromosomal
regions resulted from small mutations such as frame
shifts or gene duplication events in a single strain.
3.5.1. K7 genes absent in S288C The genes predicted as present in K7 that are absent in S288C arelisted in Supplementary Table S5. Several K7 genes
already demonstrated involvement in the characteristic features of sake yeast, including K7_AWA1(K07_06182) and a paralogous set of K7_BIO6
genes (K7_BIO6-1/BIO6-2a/BIO6-2b/BIO6-3/BIO64a/BIO6-4b: K07_11198/11203/11204/03384/
11206/11207), which are unique to sake yeast
strains.10,11 BIO1, which is absent in S288C, is also
required for biotin biosynthesis in yeast.44 As expectedfrom the biotin prototrophy displayed by K7, BIO1
orthologs were also found in K7 (K7_BIO1-1/BIO12/BIO1-3: K07_00624/03376/03381).11,45
Three paralogous genes that are not found in S288C,
K07_00009/11194/04100 (named K7_EHL1/EHL2/
EHL3 in this study), were predicted to encode proteins
similar to bacterial epoxide hydrolase. Numerous

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also proposed for the diploid strain YJM128.41
However, LOH in K7 was far more extensive thanthat found in YJM128, resulting in 82.7% of theentire genome being almost homologous. As LOH ismainly caused by mitotic recombination, the probability of LOH events is dependent on the number ofclonal generations during asexual proliferation.
Therefore, it is likely that K7 passed through more
mitotic generations than YJM128 after the out-crossing event. A sporulation defect of K7 may have contributed to a long-term clonal proliferation thatallowed extensive LOH events without meiosis. LOH
events also result in the selection of one haplotype
between two homologous chromosomes whose haplotypes differ from each other. Consequently, LOH
can be a major driving force in the diversificationand microevolution of diploid strains, such as K7,
which lost a meiotic life cycle, as observed in
Candida albicans.42[Vol. 18,

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3.5.2. S288C genes absent in K7 Forty-nine
genes predicted as present in S288C but absent inK7 are listed in Supplementary Table S6. We confirmed that these genes are not present in K7 nonsupercontigs. Notably, two subtelomeric paralogous
blocks in the S288C genome, containing HXT15 –SOR2 – MPH2 on chromosome IV and HXT16–
SOR1 – MPH3 on chromosome X, were not identified
in the K7 genome. The paralogous pairs of HXT15
and HXT16, SOR1 and SOR2 and MPH2 and MPH3
encode nearly identical hexose transporters, sorbitol
dehydrogenases and maltose transporters, respectively.46 – 48 It is likely that non-reciprocal chromosomal recombinations in subtelomeric regions caused
duplication of these sequences in S288C, but resulted
in their loss in the K7 lineage. Another subtelomericgene, AIF1, which is located on S288C chromosome
XIV and encodes a mitochondrial cell death factor,49
was also lost in K7. CWP1, encoding a cell-wallprotein linked to glucan chains,50 was disrupted bya frame-shift mutation in K7. Although the effects of
the loss of this protein are unclear, it is possible thatthe cell-wall properties of K7 are affected.
PPT1, which encodes a protein phosphatase,51 is
located at an internal region of chromosome VII in
S288C, whereas the corresponding 2.6-kb region
was lost and replaced with a Ty element(K7_YGRCTy2-2) in the K7 genome (Fig. 4A). The
effect of the loss of PPT1 on cellular function or thesake brewing character of K7 is unclear. A tRNA
gene tR(UCU)G3 on the left side of PPT1 was alsoabsent in K7. However, the tI(AAU)G gene locatedon the right side of PPT1 in S288C was present in
K7. Sequences located several hundred bases
upstream of a tRNA gene can serve as potential
target sites for Ty integration. Consequently, multiple
Ty insertion and subsequent excision events may haveresulted in the two solo LTRs on both sides of PPT1 in a
K7 ancestral genome. Recombination, including insertion or gene conversion, between a Ty cDNA and yeast
chromosome with LTRs, was reported in laboratory
experiments.52 Therefore, we speculate that geneconversion between a double-stranded Ty cDNA andtwo solo LTRs pre-existing on the chromosome VII
may have occurred in the K7 ancestor, resulting in
the replacement of PPT1 with Ty2 (Fig. 4,
Supplementary Fig. S6). Alternatively, double reciprocal crossing-over may have resulted in the observed
structure; however, this is unlikely, as PPT1 was notfound in either the supercontigs or the non-supercontig contigs of K7. A similar structure was also observedin the K7 right arm of chromosome I (Fig. 4B), involving the replacement of two tRNA genes tL(CAA)A
and tS(AGA)A with Ty2, which we speculate may
have involved a similar gene conversion mechanism.
In previous studies, Ty cDNA-mediated gene conversions were only observed in genetically modified
strains and were thought to be an artificiallyinduced phenomena.52 The structures identified in
the K7 genome would represent the first example ofthe spontaneous direct Ty-mediated gene conversion
in a wild-type strain.
Two tandemly-duplicated acid phosphatase genes,
PHO3 and PHO5, are located on chromosome II of
the S288C genome.53 However, K7 possessed only
PHO5 (K07_00381), consistent with the observationthat K7 shows repressive, but not constitutive, acidphosphatase activity.54 These tandemly arranged
acid phosphatase genes are also present in YJM789,
EC1118andseveralother
Saccharomyces
species.15,17,55 The prevalence of these genes suggeststhat PHO3 was looped out of the K7 genome by homologous recombination. In the K7 genome, no copies
of ASP3 and its neighboring ORFs, which encode
cell-wall L-asparaginase II and putative proteins of
unknown function, were found, even though S288C

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bacterial orthologs to K7_EHL1/EHL2/EHL3 have been
identified in the DDBJ/EMBL/GenBank non-redundant
database, whereas eukaryotic orthologs have only
been found in S. paradoxus to date (Supplementary Fig.
S4). Thus, these epoxide hydrolase genes may havebeen horizontally transferred from bacteria to acommon ancestor of these yeasts. K7_EHL1/EHL2/
EHL3 should be involved in the detoxification ofharmful epoxide compounds. However, the actual substrates are unknown, and to date, no epoxide compounds have been identified from sake or fermenting
sake mash.
K7_02354, which was located in a subtelomericregion, is orthologous to YJM-GNAT of YJM789,
which encodes a gene similar to bacterial GCN5related N-acetyltransferase, suggesting that acommon ancestor of K7 and YJM789 acquired theirancestral gene by horizontal transfer.15 Sequences ofK7_02354 and YJM-GNAT were almost identical(over 99%) at both the nucleotide and the aminoacid levels (Supplementary Fig. S5).
K7_KHR1 (K07_03550), which encodes a previously identified heat-resistant killer toxin,45 was
located in an internal region of chromosome IX andwedged between two solo LTRs. Although a similar
structure was observed in the EC1118 genome,17
S288C does not possess KHR1, and only a solo LTR(YILCdelta3) is located in the corresponding locus.
This suggests that the loss of KHR1 in S288C wascaused by LTR-mediated recombination, as predicted
in a previous study.17 A large proportion of genes
unique to K7 (Supplementary Table S5) have notbeen characterized, and their involvement and function in the characteristic features of sake yeast
remain to be explored.

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Genome Sequencing of Sake Yeast[Vol. 18,

chromosome XII contains at least four copies of ASP3
adjacent to rDNA repeats.56 These sequences arealso absent in numerous other S. cerevisiae
strains.15 – 17,39 Although tandemly triplicated ENA1/
ENA2/ENA5, encoding P-type ATPases are located on
chromosome IV in S288C,57 only one copy was identified in K7 (K7_ENA1/K7_01190), as observed inmany other strains.15 – 17,39 The triplication is a relatively specific feature of S288C and its related
strains. The K7 genome also contained one copy of
CUP1 (K7_CUP1/K07_03116), encoding a metallothionein, whereas S288C contains two copies intandem,58 consistent with the lower copper resistanceof K7 than that of X2180, an isogenic diploid of
S288C (data not shown).
3.6. Hexose transporter and GAL genes
Transport of sugars across the membrane is one of
the key steps in ethanol fermentation. The S288C
genome contains 20 genes that encode hexose transporter family proteins (HXT1 –HXT17 and GAL2) andglucose sensors (SNF3 and RGT2) as shown in Fig. 5.
The products of these genes show different glucose
affinities and are differentially expressed in order tocoordinately control glucose uptake in environmentswith a broad range of glucose concentrations.59
Many of these genes, including two glucose sensors,
were highly conserved among S288C and K7. In particular, both the low-affinity glucose transporter
genes HXT1/HXT3 were conserved, implying thatthey may be responsible for glucose uptake during
the sake brewing process, as reported in wine
yeast.60 HXT5/HXT6/HXT7 were located at contig
ends, and the DNA sequences of these regions in K7
were not completely analyzed. In S288C, HXT6 andHXT7 encode nearly identical proteins and arearranged in tandem; however, they may have beencombined into a chimeric gene (HXT6/7) in K7, as
observed in other strains.59
We revealed that two distinct classes of S288C HXT
genes displayed almost altered gene structures in K7.
The first contains HXT9/HXT11 and HXT12, which isannotated as a possible pseudogene due to a frameshift mutation in SGD. In the K7 genome, although oneof the duplicated HXT9 orthologs was conserved(K7_HXT9-2/K07_02205: 98% identical to HXT9 at
the amino acid level), all other HXT9/HXT11 orthologs
were divide into two ORFs due to frame shifts

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Figure 4. Loss of genomic regions by Ty2 replacement in the K7 genome. Each region within the dotted-line box in the S288C genome was
replaced by a Ty element (Ty2) in the K7 genome. (A) A 2628-bp region containing the tRNA gene tR(UCU)G3 and PPT1/YGR123C in
S288C chromosome VII was replaced by K7_YGRCTy2-2 in K7 chromosome VII. (B) A 2464-bp region containing two tRNA genes,tL(CAA)A and tS(AGA)A, in S288C chromosome I was replaced by K7_YARWTy2-1 in K7 chromosome I.

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T. Akao et al.

431

Figure 5. Phylogenetic tree of hexose transporter family proteins
identified in S288C. The amino acid sequences of S288C HXT
gene products were clustered by CLUSTALW 1.83, and thedendrogram was plotted using the TreeView program. Symbols
at the end of the branches indicate the structural class of K7
orthologous gene products, as shown in the box under thedendrogram. Two groups of Hxts, whose functional structures
were disrupted in K7, are framed with rectangles. Underlines
indicate genes whose expression is repressed under highglucose conditions. The letter (a) denotes each of the two K7
orthologs is indicated as a half round.

(K7_HXT9-1a/HXT9-1b/HXT11a/HXT11b: K07_03662/
03663/06179/06180). The disrupted structure of
HXT12 was also conserved in K7 (K7_HXT12a/HXT12b:
K07_03394/03395) as well as in S288C(Supplementary Table S4). Four genes HXT13/
HXT15/HXT16/HXT17 are classified into the second
HXT class. In the K7 genome, the HXT13/HXT17
orthologs (K7_HXT13a/HXT13b/HXT17a/HXT17b:
K07_01805/1807/06162/
06164)
contained
frame-shift mutations that may have resulted in theloss of function (Supplementary Table S4), while thesequences corresponding to HXT15/HXT16 wereabsent in the K7 genome (Supplementary Table S6).
Thus, in K7, the functions of gene products in thisclass appeared to be completely lost, although theirmolecular functions were unclear.
In the K7 genome, we also identified frame-shift
mutations in the GAL3 and GAL4 genes that divided

3.7. Structure of the HO gene
As K7 is a heterothallic diploid, HO involved inmating-type switching was predicted as nonfunctional in K7.63 Indeed, a homozygous mutationof A1424T (H475L), which was reported to cause aloss of function in S288C,64 was observed in the K7
ho allele. Moreover, a 36-amino acid deletion at524 – 559 was also present in K7, as was reported in
the heterothallic bioethanol strain JAY291.18
Collectively, these differences are likely to be responsible for the observed K7 heterothallism.
3.8. Comparison of Ty elements and LTRs
The chromosomal insertion of a Ty element can
result not only in the loss or alteration of gene function,
but may also modify gene expression levels. The annotated Ty elements and solo LTRs from K7 are summarized in Supplementary Tables S7 and S8. Nearly all Ty
and solo LTR insertions followed the target-site-selection rule, displaying preferential insertion within a 1-kb
upstream region of RNA polymerase III-target genes(Supplementary Table S8). We compared Ty insertion
events, which included intact Ty elements and solo
LTRs, between K7 and S288C. If flanking regions of
two Ty elements, a Ty element and a solo LTR or two
solo LTRs, were identical between the two strains, weestimated that these insertion sequences should bederived from the same Ty element insertion event in
the common ancestral strain. A total of 198 Ty insertion events were estimated to be identical between
K7 and S288C, while 121 and 137 insertion eventswere unique to K7 and S288C, respectively(Supplementary Tables S7B and S8). Interestingly,
among the same 198 insertion events, only one pair

Downloaded from https://academic.oup.com/dnaresearch/article-abstract/18/6/423/477017 by guest on 17 June 2019each gene into two ORFs (K07_01156 andK07_01157 for GAL3 and K07_06847 andK07_06846 for GAL4). Thus, both Gal3p and Gal4p
in K7 are shorter at their C termini than their orthologs in S288C, suggesting that their molecular functions may be impaired. Since Gal3p and Gal4p
function as an inducer and activator, respectively,
which constitute the transcriptional induction
system of galactose assimilating genes,61 the loss ofGal3p and Gal4p functions may lead to defective
GAL gene induction in response to galactose.
Consistent with this speculation, the assimilationand fermentation of galactose are remarkably weakened in several sake yeast strains, including K7.62
Our analyses suggest that K7 possesses different
sugar uptake and assimilation properties from thoseof S288C. It is likely that sake yeast cells could tolerate
a functional loss from these genes without a negative
selective force due to the growth of these strains in
the glucose-rich environment specific to sake mash.

432

Genome Sequencing of Sake Yeast

4.

Conclusions

We revealed the sequence and structure of the K7
genome that represents the first such study of a sakeyeast lineage within S. cerevisiae and provides thebasis for future studies on the brewing characteristics,
genealogy and evolution of sake yeast. The phenotypic
effects of the identified structural polymorphisms
between K7 and S288C genomes are largely
unknown and remain to be explored. In addition,
the uneven heterozygosity distribution found in theK7 genome is suggestive of the microevolution of K7
and related sake yeast strains. Future genetic studies
encompassing a wide range of Saccharomyces strains
are necessary to resolve the genetic basis for thecharacteristics and evolution of sake yeast strains ingreater detail.
The sequences and annotations reported in this paper
have been deposited at DDBJ/EMBL/GenBank under theaccession no. BABQ01000001–BABQ01000705,
DG000037–DG000052 and AP012028. Informationof the sequence and gene annotation are also available
on the sake yeast genome database (http://nribf1.nrib.
go.jp/SYGD/) and database of the genomes analyzed
at NITE (DOGAN; http://www.bio.nite.go.jp/dogan/top/).
Acknowledgements: The authors thank Tokio
Ichimatsu, Hikaru Suenaga (Fukuoka Industrial
Technology Center), Takafumi Kubodera, Hiroyuki
Senju, Nobuo Yamashita (Hakutsuru Sake Brewing
Co., Ltd, Nihonsakari Co., Ltd), Hideki Hirakawa(Kazusa DNA Research Institute), Yasuyuki Masuda,
Tasuku Yamada, Yasuhiko Wada (Kiku-Masamune Sake
Brewing Co., Ltd), Shigehito Ikushima (Kirin Holdings
Co., Ltd), Kenichiro Hara, Jo Kuwashima (NihonsakariCo., Ltd), Yoshinobu Kaneko, Minetaka Sugiyama(Osaka University), Takayuki Bogaki, Akio Koda (Ozeki
Corporation), Naoyuki Kobayashi (Sapporo BreweriesLtd), Takahiro Oura (Tokyo Institute of Technology),
Hiroyuki Horiuchi, Ryoichi Fukuda and Jun-ichi
Maruyama (University of Tokyo) for their contributions
in gene annotation and Taku Kato, Naoki Maeya andChie Oishi (National Research Institute of Brewing) for
their contribution in validating the heterozygosity
positions.
Supplementary data: Supplementary data are
available at www.dnaresearch.oxfordjournals.org.

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