U.S. patent application number 10/791791 was filed with the patent office on 2004-12-30 for screening method for genes of brewing yeast.
This patent application is currently assigned to SUNTORY LIMITED. Invention is credited to Ashikari, Toshihiko, Fujimura, Tomoko, Kodama, Yukiko, Nakamura, Norihisa, Nakao, Yoshihiro.
Application Number | 20040265862 10/791791 |
Document ID | / |
Family ID | 32958746 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265862 |
Kind Code |
A1 |
Nakao, Yoshihiro ; et
al. |
December 30, 2004 |
Screening method for genes of brewing yeast
Abstract
The present invention provides (A) a method for the selection of
genes participating in the desired brewing character, which
comprises preparing a database compiling the data of the whole
genome sequence of industrial yeast, particularly a brewing yeast
used for alcoholic beverages; selecting gene participating in a
desired brewing character that the brewing yeast specifically
possesses; and carrying out functional analysis of the gene by
disruption or overexpression; (B) a DNA array in which
oligonucleotide(s) selected based on the data base compiling the
data of the whole genome sequences of an industrial yeast, (C) a
breeding method for constructing improved cultures achieving the
desired brewing character, (D) a method for producing an alcohol or
an alcoholic beverage in which productivity and quality are
improved using the yeast, (E) a gene which is specific to the
improved brewing yeast, and (F) a peptide encoded by the gene.
Inventors: |
Nakao, Yoshihiro;
(Kyoto-shi, JP) ; Nakamura, Norihisa; (Kyoto-shi,
JP) ; Kodama, Yukiko; (Osaka, JP) ; Fujimura,
Tomoko; (Osaka, JP) ; Ashikari, Toshihiko;
(Osaka, JP) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
SUNTORY LIMITED
OSAKA
JP
|
Family ID: |
32958746 |
Appl. No.: |
10/791791 |
Filed: |
March 4, 2004 |
Current U.S.
Class: |
506/9 ;
435/6.12 |
Current CPC
Class: |
C12C 12/006 20130101;
C07K 14/395 20130101; C12N 1/18 20130101; C12Q 1/6895 20130101;
Y02E 50/10 20130101; C12C 12/004 20130101; C12P 7/06 20130101; C12N
15/1034 20130101; Y02E 50/17 20130101; C12Q 2600/156 20130101; C12Q
2600/158 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2003 |
JP |
057677/2003 |
Claims
1. A screening method for genes participating in increase in
productivity and/or improvement in flavor in the production of an
alcohol or an alcoholic beverage, characterized in that, (a) the
whole genome sequence of industrial yeast is analyzed, (b) the
sequence is compared with that of Saccharomyces cerevisiae, (c) a
gene of the industrial yeast encoding an amino acid sequence having
70 to 97% identity to an amino acid sequence encoded by the gene of
Saccharomyces cerevisiae is selected, and (d) functional analysis
of the selected gene is carried out, whereby the character given to
the yeast by the gene is identified.
2. The method according to claim 1, wherein a DNA array is used for
the functional analysis in (d).
3. The method according to claim 2, wherein said DNA array
comprises one or more of oligonucleotides adhered to a solid
support; said one or more oligonucleotides comprise a DNA sequence
having 10 to 30 nucleotides existing in an open reading frame of
the whole genome sequence of an industrial yeast and not existing
in the region other than the region of said 10 to 30 nucleotides
sequence in the whole genome sequence, or its complementary DNA
sequence.
4. The method according to claim 3, wherein said one or more of
oligonucleotides are hybrized under a stringent condition.
5. The method according to claim 2, wherein said DNA array,
comprises one or more oligonucleotides adhered to a solid support;
said one or more oligonucleotides comprise a DNA sequence having 10
to 30 nucleotides existing in a non-coding region of the whole
genome sequence of an industrial yeast and not existing in the
region other than the region of said 10 to 30 nucleotides sequence
in the whole genome sequence, or its complementary DNA
sequence.
6. The method according to claim 5, wherein said one or more of
oligonucleotides are hybridized under a stringent condition.
7. The method according to claim 3, wherein said DNA array
comprises two or more oligonucleotides.
8. The method according to claim 1, wherein the industrial yeast is
brewing yeast.
9. The method according to claim 1, wherein the brewing yeast is
beer yeast.
10. A gene which is obtained by the screening method according to
claim 1.
11. The gene according to claim 10, which is characterized by being
able to increase the concentration of sulfite in a culture medium
of yeast when said gene is expressed in yeast.
12. DNA which comprises a DNA sequence represented by SEQ ID NO: 1
or 2, and DNA which hybridizes to said DNA under stringent
condition.
13. DNA which encodes a polypeptide having an amino acid sequence
represented by SEQ ID NO: 3 or 4, and DNA which encodes polypeptide
having an amino acid sequence in which one to several amino acid
residues are deficient, substituted, added or a combination thereof
in an amino acid sequence represented by SEQ ID NO: 3 or 4.
14. A recombinant vector containing the gene of claim 10.
15. The recombinant vector according to claim 14, wherein a
promoter, a terminator, or both are placed adjacent to said
gene.
16. The recombinant vector according to claim 15, wherein said
promoter shows constitutive expression.
17. The recombinant vector according to claim 15, wherein said
promoter is a promoter of glyceraldehyde-3-phosphate dehydrogenase
gene.
18. A transformant containing the gene according to claim 10.
19. The transformant according to claim 18, wherein said
transformant belongs to yeast of genus Saccharomyces.
20. A polypeptide encoded by the gene according to claim 10 or a
polypeptide having an amino acid sequence in which one to several
amino acid residues are deficient, substituted, added, or a
combination thereof in an amino acid sequence in the said
polypeptide.
21. A polypeptide having an amino acid sequence represented by SEQ
ID NO: 3 or 4 or a polypeptide having an amino acid sequence in
which one to several amino acid residues are deficient,
substituted, added, or a combination thereof in the amino acid
sequence represented by SEQ ID NO: 3 or 4.
22. A method for the production of an alcohol or an alcoholic
beverage comprising subjecting the transformant according to claim
18 to fermentation in a sugar-containing medium selected from the
group consisting of wort, grape juice, rice juice and glucose
syrup.
23. A breeding method of yeast which is suitable for the production
of an alcohol or an alcoholic beverage comprising controlling
expression of the gene according to claim 10.
24. The breeding method according to claim 23, wherein the yeast
belongs to the genus Saccharomyces.
25. Yeast obtained by the breeding method according to claim
23.
26. A method for the production of an alcohol or an alcoholic
beverage comprising using the yeast according to claim 25.
27. An alcohol or an alcoholic beverage which is produced using the
method for the production of an alcohol or an alcoholic beverage
according to claim 26.
28. A DNA array comprising one or more oligonucleotides adhered to
a solid support: said one or more oligonucleotides comprise a DNA
sequence having 10 to 30 nucleotides existing in an open reading
frame of the whole genome sequence of an industrial yeast and not
existing in the region other than the region of said 10 to 30
nucleotides sequence in the whole genome sequence, or its
complementary DNA sequence.
29. The DNA array according to claim 28, wherein said one or more
of oligonucleotides are hybridized under a stringent condition.
30. A DNA array comprising one or more oligonuclaeotides adhered to
a solid support: said one or more oligonucleotides comprise a DNA
sequence having 10 to 30 nucleotides existing in a non-coding
region of the whole genome sequence of an industrial yeast and not
existing in the region other than the region of said 10 to 30
nucleotides sequence in the whole genome sequence, or its
complementarty DNA sequence.
31. The DNA array according to claim 30, wherein said one or more
of oligonucleotides are hybridized under a stringent condition.
32. The DNA array according to claim 28, wherein said DNA array
comprises two or more oligonucleotides.
33. The method according to claim 5, wherein said DNA array
comprises two or more oligonucleotides.
34. A recombinant vector containing the DNA of claim 11.
35. The recombinant vector according to claim 34, wherein a
promoter, a terminator, or both are placed adjacent to said
DNA.
36. The recombinant vector according to claim 35, wherein said
promoter shows constitutive expression.
37. The recombinant vector according to claim 35, wherein said
promoter is a promoter of glyceraldehyde-3-phosphate dehydrogenase
gene.
38. A recombinant vector containing the DNA of claim 12.
39. The recombinant vector according to claim 38, wherein a
promoter, a terminator, or both are placed adjacent to said
DNA.
40. The recombinant vector according to claim 39, wherein said
promoter shows constitutive expression.
41. The recombinant vector according to claim 39, wherein said
promoter is a promoter of glyceraldehyde-3-phosphate dehydrogenase
gene.
42. A transformant containing the DNA according to claim 12.
43. The transformant according to claim 42, wherein said
transformant belongs to yeast of genus Saccharomyces.
44. A transformant containing the DNA according to claim 13.
45. The transformant according to claim 44, wherein said
transformant belongs to yeast of genus Saccharomyces.
46. The DNA array according to claim 30, wherein said DNA array
comprises two or more oligonucleotides.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a screening method for
genes of an industrial yeast used for the production of an
alcoholic beverage such as beer or sake, a fuel alcohol, etc. and
particularly for genes of brewing yeast used for the production of
an alcoholic beverage. More particularly, it relates to a method
where, in the production of an alcoholic beverage, DNA sequence
information of brewing yeast is compiled in a database so that the
gene which participates in increase in productivity and/or
improvement in flavor such as stabilization, reinforcement, etc. of
the flavor is selected; a method for breeding yeast suitable for
the brewing in which expression of a gene is controlled, such as
yeast in which the selected gene is disrupted or yeast in which the
gene is overexpressed; and a method for the production of an
alcoholic beverage using the bred yeast.
[0003] 2. Description of the Prior Art
[0004] Development of techniques for production of fuel alcohols,
alcoholic beverages such as beer or sake, etc. has been carried out
using industrial yeast. Especially in the production of an
alcoholic beverage using brewing yeast, there has seen a brisk
development in the techniques for increasing productivity and
improving flavor such as stabilization or enhancement of flavor of
an alcoholic beverage.
[0005] The most consumed alcoholic beverage in the world is beer
and the amount of beer produced in the world in 2001 was about
140,000,000 kL. Type of beer is roughly classified into three
depending upon type of yeast and fermentation method. The three
types are, naturally fermented beer where fermentation is carried
out utilizing yeast and microorganisms inhabiting in breweries;
ale-type beer where fermentation is carried out using a top
fermenting yeast belonging to Saccharomyces cerevisiae
(hereinafter, abbreviated as S. cerevisiae) at the temperature of
20 to 25.degree. C. and the following aging period is shortened;
and lager-type beer where fermentation is carried out using a
bottom fermenting yeast belonging to Saccharomyces pastorianus at
the temperature of 6 to 15.degree. C. and then subjected to a
low-temperature aging. At present, not less than 90% of the beer
produced in the world is a lager-type beer and, therefore, the
bottom fermenting yeast that is used for brewing of the lager-type
beer has been most widely used in beer brewing.
[0006] In the so-called fermentation production where production is
carried out using a microorganism including the above-mentioned
brewing yeast, it is important that the fermentation process is
optimized and that the useful strain is selected and bred, in order
to increase productivity and improve quality of the product.
[0007] In the case of optimization of beer brewing, there has been
conducted a method where an amount of yeast metabolites such as
alcohol (e.g. ethanol), ester, organic acid, etc. are monitored,
and then temperature, quantity of airflow, content of raw material,
etc. are controlled. In such a case, material uptake and excretion
by yeast cells and metabolism in the cells are handled as a black
box and only very superficial control is carried out. In addition,
for the purpose of giving, for example, high flavor to an alcoholic
beverage, a process control method for suppressing the amount of
oxygen supply during beer brewing or the like has been tried. In
such a method, however, growth rate of the yeast itself is reduced
due to insufficient oxygen, and adverse effect such as retardation
of fermentation and/or deterioration of beer quality may arise.
Accordingly, there has been a limit on the improvement in
productivity and quality of beer by means of optimization of
fermentation processes.
[0008] On the other hand, with regard to a method of breeding
useful industrial yeast such as useful beer yeast, a technique for
selecting desirable strain has been widely used rather than actual
breeding. Beer brewing per se has been performed since well before
the discovery of microorganisms by Pasteur and, in the beer
brewing, a method of selecting more suitable strain of beer yeast
from many strains of yeast used in the beer brewery has been
traditionally carried out while there have been few cases where
beer yeast with good traits is positively bred.
[0009] As an example of a positive breeding method, there is a
method where artificial mutagenesis by chemicals or radioactive
rays is used. However, brewing yeast, particularly a bottom
fermenting yeast which is widely used in beer brewing, is in many
cases a polyploid. In that case, it is not possible to produce the
desired mutant unless mutation takes place in all of the alleles to
be mutated. Accordingly, although it is possible to induce
desirable mutation in the case of a haploid laboratory yeast, it is
substantially impossible in the case of beer yeast which is a
polyploid.
[0010] In recent years, there has been tried a breeding where
mutation or cross-breeding is carried out by using spores isolated
from bottom fermenting yeast (c.f., for example, Non-Patent
Document 1). However, the bottom fermenting yeast is a polyploid,
and has complicated chromosome structure, therefore, isolation of
spores having proliferation ability is difficult, and moreover it
is almost impossible to obtain a strain with good traits
therefrom.
[0011] On the other hand, it has recently become possible that
desired genes are introduced and expressed in the brewing yeast
using a genetic engineering technique, whereby it has become
possible to breed yeast with the desired character by using the
results of functional analysis of genes and the genes which have
been functionally analyzed. However, as compared with the baker's
yeast (S. cerevisiae; c.f., for example, Non-Patent Document 2) of
which the whole genome sequence is already clarified, the whole
genome sequence of the bottom fermenting yeast has not been
clarified and there have been only a very few findings about the
gene participating in brewing character specific to bottom
fermenting yeast and about the function of the said gene in beer
brewing.
[0012] In recent years, transcriptome analysis has been conducted
using DNA microarray where DNA fragments or nucleotide oligomers,
each of which has a partial sequence of structural gene or internal
region of the chromosome are fixed on solid support. For example,
Olesen, et al. conducted a comprehensive genetic expression
analysis of bottom fermenting yeast during the brewing using
GeneFilters (manufactured by Research Genetics Co.) (c.f., for
example, Non-Patent Document 3). However, since the whole genome
sequence of bottom fermenting yeast has not been clarified yet, it
is ambiguous that what gene is monitored for its expression
precisely. As a result, such information is quite insufficient to
apply to metabolic analysis of bottom fermenting yeast, and to
breeding of useful yeast, and to control of beer brewing
process.
[0013] At present, the whole genome sequences of more than 100
species of microorganisms have been determined (c.f., for example,
Non-Patent Document 6) including S. cerevisiae, Escherichia coli
(c.f., for example, Non-Patent Document 4) and Mycobacterium
tuberculosis (c.f., for example, Non-Patent Document 5). On the
basis of the determined DNA sequences, genes of these
microorganisms are identified and function of an enormous number of
genes have been predicted without conducting genetic, biochemical
and molecular biological experiments. However, industrial yeast
such as brewing yeast which has high ploidy and complicated
chromosome structure, and thus an assembly (an operation for
connecting the DNA sequences) is presumed to be difficult.
Therefore, the whole genome sequence of bottom fermenting yeast
which contains two different types of genome (c.f., for example,
Non-Patent Document 7) has not been reported yet.
[0014] In the production of specific alcohols or alcoholic
beverages, there is a technique to increase concentration of
sulfite in the product for control of flavor. Sulfite is known as a
compound which has anti-oxidative activity, and has been widely
used as an antioxidant in the fields of food, beverage and
pharmaceuticals, and also in an alcoholic beverage. For example, in
the case of wine that requires a long aging period, sulfite plays
an important role for the preservation of its quality. It is also
known that, in beer brewing, the quality preservation period
becomes long in accordance with the increase in concentration of
sulfite contained in the product. Thus, when the amount of sulfite
in the product is increased, it is possible to prepare a product
that has excellent flavor stability and a long quality preservation
period.
[0015] The simplest way to increase the amount of sulfite in the
product is addition of sulfite. In Japan, so far as wine is
concerned, it is permitted by the Ministry of Health, Labor and
Welfare to add sulfite to an extent of not more than 350 ppm in
terms of residual sulfite concentration. In that case, however,
since sulfite is categorized as food additives, it is not
appropriate to add sulfite to beer when a negative image of
consumers to food additives is taken into consideration.
[0016] However, the yeast used in brewing produces hydrogen sulfide
by the reduction of sulfate in the medium in order to synthesize
sulfur-containing metabolites such as sulfur-containing amino
acids. Sulfite is an intermediate metabolite of this pathway. If
sulfite is efficiently excreted outside the cells during
fermentation period, the amount of sulfite both in the wort and in
the product can be increased.
[0017] There are two methods for increasing sulfite concentration
in the wort during fermentation. One is control of fermentation
process and another is breeding of brewing yeast. As for control of
fermentation process, amount of sulfite produced during
fermentation is inversely proportional to the concentration of
dissolved oxygen and, therefore, there has attempted, a method
where the concentration of dissolved oxygen is reduced so that
amount of sulfite is increased and at the same time the oxidation
of sulfite is suppressed. However, in that method, growth rate of
yeast is reduced due to lack of oxygen, which has negative effects
such as retardation of fermentation and deterioration of quality.
Therefore that method is not practical.
[0018] On the other hand, as mentioned above, a genetic engineering
technique has been developed for breeding brewing yeast. For
example, there are some reports focused on sulfur metabolism of
yeast. Sulfite (SO.sub.2) is an intermediate product of
sulfur-containing amino acid and vitamin synthesis and is produced
via a pathway of sulfate ion (SO.sub.4.sup.2-).fwdarw.APS (adenyl
sulfate) PAPS (phosphoadenylyl sulfate) sulfite ion
(SO.sub.3.sup.2-) where the sulfate ion is incorporated from
outside of the cells. There is an attempt that copy numbers of MET
3 gene participating in the reaction of sulfate ion
(SO.sub.4.sup.2-).fwdarw.APS (adenylyl sulfate) and of MET 14 gene
participating in the reaction of APS (adenylyl sulfate).fwdarw.PAPS
(phosphoadenylyl sulfate) are increased to improve the ability of
the yeast for the production of sulfite (c.f., for example,
Non-Patent Document 8). There is another example of an attempt
where reduction of sulfite ion (SO.sub.3.sup.2-) is inhibited by
disruption of MET 10 gene whereby amount of sulfite produced by the
yeast is increased (c.f., for example, Non-Patent Document 9).
According to such attempts, amount of sulfite produced by an MET 10
gene disruptant is increased to an extent of not less than ten-fold
of the parental strain, but on the other hand, some retardation in
fermentation and increase in the amounts of acetaldehyde and
1-propanol in the beer are noted, which has become a problem for
the practical use.
[0019] Accordingly, although development of breeding methods for
industrial yeast such as brewing yeast using genetic engineering
have been in progress, it is the current status that, due to
insufficient genomic information of brewing yeast, selection of the
gene participating in a brewing character of brewing yeast,
analysis of function of protein encoded by the gene and utilization
of those findings for breeding have not been sufficiently carried
out.
[0020] Thus, a method for breeding yeast which shows the desired
character without deterioration of fermentation speed and product
quality has not been established yet and there has been a big
demand for the development of such a method not only in the brewing
industry but also in the industries where yeast is used.
[0021] (Non-Patent Document 1) C. Gjermansen: "Construction of a
hybrid brewing strain of Saccharomyces carlsbergensis by mating of
meiotic segregants", Carlsberg Res. Commun., volume 46, pages 1 to
11 (1981).
[0022] (Non-Patent Document 2) A. Goffeau, et al.: "The Yeast
Genome Directory", Nature, volume 387, pages 5 to 105 (1997).
[0023] (Non-Patent Document 3) K. Olesen, et al.: "The dynamics of
the Saccharomyces carlsbergensis brewing yeast transcriptome during
a production-scale lager beer fermentation", FEMS Yeast Research,
volume 2, pages 563 to 573 (2000).
[0024] (Non-Patent Document 4) F. R. Blattner, et al.: "The
Complete Genome Sequence of Escherichia coli K-12", Science, volume
277, pages 1453-1462 (1997).
[0025] (Non-Patent Document 5) S. T. Cole, et al.; "Deciphering the
biology of Mycobacterium tuberculosis from the complete genome
sequence", Nature, volume 393, pages 537-544 (1998).
[0026] (Non-Patent Document 6) The National Center for
Biotechnology Information,
http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html.
[0027] (Non-Patent Document 7) Y. Tamai et al.: "Co-existence of
two types of chromosome in the fermenting yeast, Sacchaomyces
cerevisiae", Yeast, volume 10, pages 923-933 (1998).
[0028] (Non-Patent Document 8) C. Korch, et al.: Proc. Eur. Brew.
Conv. Congress, Lisbon, pages 201-208 (1991).
[0029] (Non-Patent Document 9) J. Hansen, et al.: "Inactivation of
MET 10 in brewer's yeast specifically increases SO.sub.2 formation
during beer production", Nature Biotech., volume 14, pages
1587-1591 (1996).
[0030] (Non-Patent Document 10) T. Sijen, et al.: "Transcriptional
and posttranscriptional gene silencing are mechanistically
related", Curr. Biol., volume 11, pages 436-440 (2001).
[0031] (Non-Patent Document 11) N. Goto, et al.: "SSU1-R, a
sulphite resistance gene of wine yeast, is an allele of SSU 1 with
a different upstream sequence", J. Ferment. Bioeng., volume 86,
pages 427-433 (1998).
[0032] (Non-Patent Document 12) D. Avram, et al.: "SSU 1 encodes a
plasma membrane protein with a central role in a network of
proteins conferring sulfite tolerance in Saccharomyces cerevisiae",
J. Bacteriol., volume 179, pages 5971-5974 (1997).
[0033] (Non-Patent Document 13) H. Park, et al.; "SSU 1 mediates
sulphite eff lux in Saccharomyces cerevisiae", Yeast, volume 16,
pages 881-888 (2000).
SUMMARY OF THE INVENTION
[0034] An object of the present invention is to provide a method of
selecting gene participating in the desired brewing character,
which is achieved in such a manner that a database compiling the
whole genome sequence (hereinafter, may be abbreviated as genomic
DB) of industrial yeast, particularly brewing yeast used for an
alcoholic beverage such as beer, is prepared; gene that the brewing
yeast possesses is selected from the database; functional analysis
of the gene is carried out by disruption or overexpression. Another
object is to provide a breeding method of the yeast showing the
brewing character which the said gene participates in and also a
method of producing an alcohol or an alcoholic beverage where
productivity and quality are improved using the said yeast. Still
another object is to provide genes mentioned above and peptides
encoded by the said genes.
[0035] It has been known that brewing yeast widely used for
industrial purpose is a polyploid and especially, bottom fermenting
yeast is an allopolyploid which is composed of at least two kinds
of genomes. One of the genomes is thought to be a genome derived
from S. cerevisiae of which the whole genome sequence has been
clarified, while the source of another genome(s) has not been
clarified yet.
[0036] The present inventors have determined the whole genome
sequence of the bottom fermenting yeast in order to find
unidentified genes displaying essential functions for excellent
brewing. The amino acid sequences of the bottom fermenting yeast
were then compared with those registered in the genomic DB for S.
cerevisiae, and functions of proteins encoded by genes of the
brewing yeast were estimated. As a result, it has been clarified
that the genes of the bottom fermenting yeast are roughly
classified into Sc type genes showing nearly 100% amino acid
identity to S. cerevisiae and non-Sc type genes showing around 70
to 97% identify. Moreover, it has been clarified that the bottom
fermenting yeast has complicated chromosome structure consists of
Sc-type chromosomes, non-Sc-type chromosomes and Sc/non-Sc-type
chimera chromosomes. Structure of the whole chromosomes of the
bottom fermenting yeast is shown in FIG. 1. On the basis of genomic
information clarified by the present invention, the present
inventors have found such an unexpectedly complicated structure of
chromosomes, and developed a screening method for the genes of
bottom fermenting yeast. To be more specific, there has been
achieved a screening method for genes participating in brewing
characters specific to the brewing yeast, which is characterized in
that (A) the whole genome sequence of industrial yeast,
particularly bottom fermenting yeast which is one of the brewing
yeasts, is analyzed, (B) the genome sequence is compared with the
whole genome sequence of S. cerevisiae, (C) genes of the bottom
fermenting yeast encoding amino acid sequences which have 70 to 97%
identities to the amino acid sequences encoded by genes of S.
cerevisiae are selected and (D) functional analysis of the selected
genes are carried out, whereby the brewing character given to the
yeast by the genes are identified. The present inventors have
repeatedly carried out intensive investigations on the basis of
those findings and accomplished the present invention.
[0037] Thus, the present invention relates to:
[0038] (1) A screening method for genes participating in increase
in productivity and/or improvement in flavor in the production of
an alcohol or an alcoholic beverage, characterized in that, (a) the
whole genome sequence of industrial yeast is analyzed, (b) these
sequence is compared with that of Saccharomyces cerevisiae, (c)
gene of the industrial yeast encoding an amino acid sequence having
70 to 97% identity to an amino acid sequence encoded by the gene of
Saccharomyces cerevisiae is selected and (d) functional analysis of
the selected gene is carried out, whereby the character given to
the yeast by the gene is identified;
[0039] (2) A screening method according to the above (1), wherein a
DNA array is used for the functional analysis in (d) of the above
(1).
[0040] (3) A method according to the above (2), wherein a DNA
array, in which one or more of oligonucleotides comprising the
following DNA sequence or its complementary DNA sequence is adhered
to a solid support, is used;
[0041] DNA sequence (1) having 10 to 30 nucleotides existing in an
open reading frame of the whole genome sequence of an industrial
yeast and (2) not existing in the region other than the region of
said 10 to 30 nucleotides sequence in the whole genome
sequence;
[0042] (4) A method according to the above (2), wherein a DNA
array, in which one or more of oligonucleotides hybridizing in a
stringent condition to the oligonucleotides defined in the above
(3) is/are adhered to a solid support, is used;
[0043] (5) A method according to the above (2), wherein a DNA
array, in which one or more of oligonuclaeotides comprising the
following DNA sequence or its complementarty DNA sequence is
adhered to a solid support, is used;
[0044] DNA sequence (1) having 10 to 30 nucleotides existing in a
non-coding region of the whole genome sequence of an industrial
yeast and (2) not existing in the region other than the region of
said 10 to 30 nucleotides sequence in the whole genome
sequence;
[0045] (6) A method according to the above (2), wherein a DNA
array, in which one or more of oligonucleotides hybridizing in a
stringent condition to the oligonucleotides defined in the above
(5) is/are adhered to a solid support, is used;
[0046] (7) A method according to the above (2), wherein a DNA
array, in which oligonucleotides selected from two or more groups
of the following 4 groups consisting of one or more of
oligonucleotides defined in the above (3), one or more of
oligonucleotides defined in the above (4), one or more of
oligonucleotides defined in the above (5), and one or more of
oligonucleotides defined in the above (6) are adhered to a solid
support, is used;
[0047] (8) The screening method according to any of the above (1)
to (7), wherein the industrial yeast is brewing yeast;
[0048] (9) The screening method according to any of the above (1)
to (8), wherein the brewing yeast is beer yeast;
[0049] (10) Gene which is obtained by the screening method
according to the above (1);
[0050] (11) The gene according to the above (10), which is
characterized by that, when the gene mentioned in the above (10) is
expressed in yeast, the concentration of sulfite in a culture
medium of the yeast increases;
[0051] (12) DNA which comprises a DNA sequence represented by SEQ
ID NO: 1 or 2, and DNA which hybridizes to the said DNA under
stringent condition;
[0052] (13) DNA which encodes a polypeptide having an amino acid
sequence represented by SEQ ID NO: 3 or 4, and DNA which encodes
polypeptide having an amino acid sequence in which one to several
amino acid residue(s) is/are deficient and/or substituted and/or
added in an amino acid sequence represented by SEQ ID NO: 3 or
4;
[0053] (14) A recombinant vector containing the gene or the DNA
mentioned in any of the above (9) to (12);
[0054] (15) The recombinant vector according to the above (9),
wherein promoter and/or terminator are/is placed adjacent to the
gene or the DNA mentioned in any of the above (10) to (13);
[0055] (16) The recombinant vector according to the above (15),
wherein the promoter is a promoter which shows constitutive
expression;
[0056] (17) The recombinant vector according to the above (15) or
(16), wherein the promoter is a promoter of
glyceraldehyde-3-phosphate dehydrogenase gene;
[0057] (18) A transformant containing the gene or the DNA or the
recombinant vector mentioned in any of the above (10) to (17);
[0058] (19) The transformant according to the above (18), wherein
the transformant belongs to yeast of genus Saccharomyces;
[0059] (20) A polypeptide encoded by the gene or the DNA mentioned
in any of the above (10) to (13) or a polypeptide having an amino
acid sequence in which one to several amino acid residue(s) is/are
deficient and/or substituted and/or added in an amino acid sequence
in the said polypeptide;
[0060] (21) A polypeptide having an amino acid sequence represented
by SEQ ID NO: 3 or 4 or a polypeptide having an amino acid sequence
in which one to several amino acid residue(s) is/are deficient
and/or substituted and/or added in the amino acid sequence
represented by SEQ ID NO: 3 or 4;
[0061] (22) A method for the production of an alcohol or an
alcoholic beverage, characterized in that, the transformant
mentioned in the above (18) or (19) is used;
[0062] (23) A breeding method of yeast which is suitable for the
production of an alcohol or an alcoholic beverage, characterized in
that, expression of the gene mentioned in the above (10) or (11) or
gene on the DNA mentioned in the above (12) or (13) is
controlled;
[0063] (24) The breeding method according to the above (23),
wherein the yeast belongs to the genus Saccharomyces;
[0064] (25) Yeast obtained by the breeding method according to the
above (23) or (24);
[0065] (26) A method for the production of an alcohol or an
alcoholic beverage using the yeast mentioned in the above (25);
[0066] (27) An alcohol or an alcoholic beverage which is produced
using the method for the production of an alcohol or an alcoholic
beverage according to the above (26);
[0067] (28) A DNA array, in which one or more of oligonucleotides
comprising the following DNA sequence or its complementary DNA
sequence is adhered to a solid support;
[0068] DNA sequence (1) having 10 to 30 nucleotides existing in an
open reading frame of the whole genome sequence of an industrial
yeast and (2) not existing in the region other than the region of
said 10 to 30 nucleotides sequence in the whole genome
sequence;
[0069] (29) A DNA array, in which one or more of oligonucleotides
hybridizing in a stringent condition to the oligonucleotides
defined in the above (28) is/are adhered to a solid support;
[0070] (30) A DNA array, in which one or more of oligonuclaeotides
comprising the following DNA sequence or its complementarty DNA
sequence is adhered to a solid support; DNA sequence (1) having 10
to 30 nucleotides existing in a non-coding region of the whole
genome sequence of an industrial yeast and (2) not existing in the
region other than the region of said 10 to 30 nucleotides sequence
in the whole genome sequence;
[0071] (31) A DNA array, in which one or more of oligonucleotides
hybridizing in a stringent condition to the oligonucleotides
defined in the above (30) is/are adhered to a solid support;
and
[0072] (32) A DNA array, in which oligonucleotides selected from
two or more groups of the following 4 groups consisting of one or
more of oligonucleotides defined in the above (28), one or more of
oligonucleotides defined in the above (29), one or more of
oligonucleotides defined in the above (30), and one or more of
oligonucleotides defined in the above (31) are adhered to a solid
support.
BRIEF DESCRIPTION OF THE DRAWING
[0073] FIG. 1 shows total chromosome structures of bottom
fermenting yeast. A white bar represents an Sc type chromosome
while a black bar represents a non-Sc type chromosome. An ellipse
represents a centromere. Roman numerals show chromosome numbers for
the corresponding S. cerevisiae. In a drawing which shows a non-Sc
chromosome, a part marked out in black shows that ligation takes
place at the region. For example, in nonScII-nonScIV, it is shown
that nonScII and nonScIV are ligated at the part marked out in
black (300 kb).
[0074] FIG. 2 shows a distribution of identify of the DNA sequence
at both ends of 3648 cosmids prepared from the genomic DNA of
strain 34/70 with the genome sequence of S. cerevisiae. The X-axis
shows the identity to S. cerevisiae and, for example, 84% on the
X-axis shows an identity of more than 82% and not more than 84%.
The Y-axis shows the numbers of cosmid end sequences showing the
identity.
[0075] FIG. 3 shows a mapping example of cosmid and shotgun clones
to genome sequence of S. cerevisiae. {circumflex over (1)} and
{circle over (2)} show genes existing on Watson strand and Crick
strand on the chromosome XVI of S. cerevisiae, respectively.
{circle over (3)} and {circle over (4)} show Sc type and non-Sc
type DNA fragments inserted in cosmid clones, respectively. {circle
over (5)} and {circle over (6)} showSc type and non-Sc type DNA
fragments inserted in shotgun clones, respectively.
[0076] FIG. 4 shows a mapping example of contigs to the genome
sequence of S. cerevisiae. (A) is a schematic depiction of
Chromosome XVI of S. cerevisiae. (B) is a drawing where the parts
of 857 to 886 kb of the Chromosome XVI of S. cerevisiae is
enlarged. Y-axis indicates % identity of contigs against S.
cerevisiae genome sequence. X-axis indicates position of contigs
against S. cerevisiae genome sequence. Contigs (solid lines) are
connected with the forward-reverse links (dot lines) from the
shotgun and cosmid reads, respectively.
[0077] FIG. 5 shows the result of DNA microarray-based comparative
genomic hybridization. The genomic DNA of strain 34/70 was
hybridized to a DNA microarray (Affymetrix Gene Chip Yeast Genome
S98 Array) and the signal of each ORF (open reading frame) was
normalized to that of the haploid strain S288C and shown as Signal
Log Ratio (2.sup.n). Signal Log Ratios were lined following genes
order in Chromosome XVI. The non-Sc type genes do not hybridize to
this Sc type array, therefore, the points (indicated by arrows)
where the Signal Log Ratios show vigorous changes were considered
to be translocation sites.
[0078] FIG. 6 shows the structure of the Chromosome XVI of strain
34/70 deduced from DNA microarray and PCR analysis.
[0079] FIG. 7 shows the fermentation profiles of SSU1 disruptants
and parental strain (BH96). a) shows yeast growth (OD 600), b)
shows the change of apparent extract (w/w %) and c) shows sulfite
concentration (ppm).
[0080] FIG. 8 shows the fermentation profiles of SSU1 overexpressed
strains and parental strain (BH225). a) shows yeast growth (OD
600), b) shows the change of apparent extract (w/w %) and c) shows
sulfite concentration (ppm).
[0081] FIG. 9 shows the change of sulfite concentration during
fermentation using MET14 overexpressed strains and parental strains
(KN009F and FOY227).
[0082] FIG. 10 shows DNA sequences of ScSSU1 and non-ScSSU1.
[0083] FIG. 11 shows DNA sequences of ScMET14 and non-ScMET14.
[0084] FIG. 12 shows the fermentation profiles of strain 34/70.
[0085] a) shows yeast growth (OD 600) and b) shows the change of
apparent extract (w/w %).
DETAILED DESCRIPTION OF THE INVENTION
[0086] With regard to the industrial yeast in the present
invention, brewing yeast for beer, wine, sake, etc. and yeasts used
for the production of fuel alcohols are exemplified. To be more
specific, yeast of genus Saccharomyces, etc. may be listed, and in
the present invention beer yeasts such as Saccharomyces pastorianus
Weihenstephan 34/70, BH 84, NBRC 1951, NBRC 1952, NBRC 1953, NBRC
1954, etc. may be used. It is also possible to use whisky yeasts
such as S. cerevisiae NCYC 90, etc., wine yeasts such as Kyokai
wine yeast No. 1, No. 3, No. 4, etc., sake yeasts such as Kyokai
sake yeast No. 7, No. 9, etc. and the like.
[0087] The screening method for genes in accordance with the
present invention is characterized in that (A) the whole genome
sequence of industrial yeast, particularly bottom fermenting yeast
which is one of the brewing yeasts, is analyzed, (B) the genomic
DNA sequence is compared with the whole genome sequence of S.
cerevisiae, (C) gene of the bottom fermenting yeast encoding an
amino acid sequence which has 70 to 97% identity to an amino acid
sequence encoded by the gene of S. cerevisiae is selected and
further (D) functional analysis of that selected gene is carried
out, whereby the brewing character given to the yeast by the gene
is identified.
[0088] It is also possible to breed yeast having an excellent
brewing character when the gene obtained by the screening method of
the present invention is used for carrying out an expression
control in such a way that the gene is overexpressed in the yeast,
and/or the gene is disrupted. Accordingly, the gene which is
obtained by a screening method of the present invention, peptide
which is encoded by the gene, a breeding method of an industrial
yeast using the gene, yeast which is obtained by the breeding
method, and a method for the production of an alcohol or an
alcoholic beverage using the yeast are also within a scope of the
present invention.
[0089] (A) Determination of the Whole Genome Sequence of Industrial
Yeast
[0090] Determination of the whole genome sequence of an industrial
yeast includes the steps of (a) genomic DNA is prepared from yeast,
(b) shotgun library and (c) cosmid library are prepared from those
genomic DNA, (d) DNA fragments to be used for determination of DNA
sequence are prepared from those library clones, (e) DNA sequence
of the library DNA fragments is determined by a sequence reaction
and (f) the sequences of those DNA fragments are assembled to
reconstruct the whole genome DNA sequence.
[0091] There is no particular limitation for the methods used for
(a) to (f) and the method may be conducted according to the known
means, while preferred method for each of them is mentioned
below.
[0092] (a) Preparation such as extraction, purification, etc. of
the genomic DNA is preferably carried out in accordance with the
known methods, for example, in "Yeast, a practical approach (IRL
Press, 6.2.1, p. 228)" and "Seibutukagakujikkennhou, No. 39,
Experiments in Yeast Molecular Genetics (edited by Yasuharu Oshima,
Gakkai Shuppan Center, pages 84 to 85, 1996)". The specific
examples of the preferred method for the preparation of DNA are
mentioned below.
[0093] Yeast cells for the preparation of genomic DNA are cultured
by a common method. With regard to a medium, any of natural and
synthetic media may be used so far as the medium contains carbon
source, nitrogen source, inorganic salt, etc. which are able to be
metabolized by the yeast, whereby cultivation of the microorganism
can be efficiently carried out. For example, YPD medium (2% (w/w)
glucose, 1% (w/w) yeast extract and 2% (w/w) polypeptone) may be
used. With regard to a method of incubation, incubation by shaking
at about 25 to 35.degree. C. through the night is recommended.
[0094] After the cultivation, cells are recovered from the culture
medium by centrifugation. The resulting cell pellet is washed with
a washing solution. Example of the washing solution is buffer A (50
mM sodium phosphate, 25 mM EDTA and 1% (v/v)
.beta.-mercaptoethanol; pH 7.5), etc. Preparation of the genomic
DNA from the washed cells may be carried out according to a common
preparation method of genomic DNA where cell walls are lysed using
Zymolyase and SDS; protein, etc. are removed using a phenol and
phenol/chloroform solution; and genomic DNA is precipitated using
ethanol or the like. To be more specific, the following method may
be exemplified.
[0095] Cultivated cells are washed and resuspended in buffer A,
then about 5 to 10 mg of Zymolyase 100T (Seikagaku Kogyo) are added
and the mixture is gently shaken at about 25 to 40.degree. C. for
about 30 minutes to 2 hours. After the shaking, buffer containing
SDS such as buffer B (0.2 M Tris-HCl, 8 0 mM EDTA and 1% SDS; pH
9.5) is added thereto and the mixture is allowed to stand at about
60 to 70.degree. C. for about 30 minutes to lyse the cells. After
that, the cell lysate is cooled on ice, mixed with 5 M potassium
acetate and allowed to stand on ice for about 60 minutes further.
The resulting solution is centrifuged (for example, at 5,000 g for
10 minutes at 15.degree. C.) to take supernatant. The same volume
of ethanol is added to the supernatant to precipitate DNA and the
mixture is immediately centrif uged (f or example, at 5,000 g for
10 minutes at 15.degree. C.) to obtain DNA. The resulting
precipitate is washed with 70% (v/v) ethanol, subjected to natural
drying and dissolved in a solution such as TE buffer (10 mM
Tris-HCl and 1 mM EDTA; pH 8.0) to give a crude genomic DNA
solution. Cesium chloride and bisbenzimide are added to and
dissolved in the crude genomic DNA solution, the mixed solution is
subjected to an ultracentrifugal separation (for example, at
100,000 g for 17 hours at 25.degree. C.), irradiation with UV light
is conducted so that the DNA bands are visualized and the lower
band is recovered. Bisbenzimide is removed by extracting the
recovered DNA solution with isopropanol which is saturated with
cesium chloride solution, then 4-fold by volume of 0.3 M sodium
acetate are added to the recovered aqueous layer followed by mixing
and the DNA is precipitated by ethanol and recovered by
centrifugation. The recovered DNA is treated with RNase and
extracted with phenol/chloroform and DNA is purified from the
recovered aqueous layer by precipitation with ethanol again. The
precipitate recovered by centrifugation is washed with 70% (v/v)
ethanol, subjected to natural drying and dissolved in a TE buffer
to prepare the genomic DNA solution.
[0096] (b) Preparation of a Shotgun Library
[0097] As to a method for the preparation of a genomic DNA library
using the genomic DNA of yeast prepared in the above (a), a method
mentioned in "Molecular Cloning, A Laboratory Manual, Third Edition
(2001)" (hereinafter, abbreviated as "Molecular Cloning, Third
Edition") may be used and, with regard to a method for the
preparation of a shotgun library which is particularly suitable for
the determination of the whole genome sequence, the following
method may be exemplified.
[0098] A TE buffer is added to the genomic DNA prepared in (a) and
the genomic DNA is fragmented using Hydroshear (manufactured by
GeneMachines) or the like. Terminal of the genome fragment is
blunted using a DNA Blunting Kit (manufactured by Takara Shuzo) or
the like, and fractionated by means of an agarose gel
electrophoresis. Then, genome fragments of about 1.5 to 2.5 kb are
excised from the gel and a buffer for the elution of DNA such as an
MG-elution buffer (0.5 mol/L ammonium acetate, 10 mmol/L magnesium
acetate, 1 mmol/L EDTA and 0.1% SDS) or the like is added to the
gel followed by shaking at about 25 to 40.degree. C. through the
night to elute DNA. The DNA eluate is treated with
phenol/chloroform and precipitated with ethanol to give a genomic
library insert. All of the above-mentioned insert and an
appropriate vector such as pUC 18 SmaI/BAP (manufactured by
Amersham Biosciences) are subjected to ligation using T4 ligase
(manufactured by Takara Shuzo) at about 10 to 20.degree. C. for
about 20 to 50 hours. The ligation reaction product is precipitated
with ethanol and the resulting recombinant vector DNA is dissolved
in an appropriate amount of TE buffer. By means of electroporation
or the like, the recombinant vector DNA is transformed to
Escherichia coli such as an Electro Cell DH5.alpha. strain
(manufactured by Takara Shuzo). It is recommended that the
electroporation is carried out under the condition mentioned in the
attached experimental manual.
[0099] The transformants into which recombinant vector containing
the genomic DNA fragments is inserted are selected on an
appropriate selective medium. For example, when pUC 18 SmaI/BAP is
used as a vector, the transformants form white colonies on an LB
plate medium (an LB medium (10 g/L of bactotryptone, 5 g/L of yeast
extract and 10 g/L of sodium chloride; pH 7.0) which contains 1.6%
of agar) containing about 0.01 to 0.1 mg/mL of ampicillin, about
0.1 mg/mL of X-gal and about 1 mmol/L of
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) upon incubation
through the night at about 30 to 37.degree. C. and, therefore, the
selection is easy. The transformants are cultured in LB medium
containing about 0.1 mg/mL of ampicillin through the night at about
30 to 37.degree. C. using a 384-well titer plate, a 50% aqueous
solution of glycerol in the same volume as the LB is added thereto
and the mixture is stirred to give a glycerol stock. Usually, the
glycerol stock can be preserved at about -80.degree. C.
[0100] (c) Preparation of a Cosmid Library
[0101] The genomic DNA prepared in (a) is subjected to a partial
digestion using an appropriate restriction enzyme such as Sau3AI
(manufactured by Takara Shuzo). It is possible to insert the DNA
fragment digested by Sau3AI into a BamHI site of a cosmid vector
such as Super CosI vector (manufactured by Stratagene) The
treatment with the restriction enzyme and the ligation may be
carried out according to the protocol attached thereto. The ligated
product obtained by such a method is subjected to a packaging
using, for example, Gigapack III Gold (manufactured by Stratagene),
and according to the manual for the experimental procedure attached
thereto, it is introduced into Escherichia coli such as an XL1-Blue
MR strain (manufactured by Stratagene). That is spread on an LB
plate medium containing ampicillin and incubated through the night
at about 30 to 37.degree. C. to get transformants. The resultant
transformants are cultured in LB medium containing about 0.1 mg/mL
of ampicillin through the night at about 30 to 37.degree. C. using
a 96-well titer plate, a 50% aqueous solution of glycerol in the
same volume as the LB is added thereto and the mixture is stirred
to give a glycerol stock. Usually, the glycerol stock can be
preserved at about -80.degree. C.
[0102] (d) Preparation of DNA Fragment for Determination of DNA
Sequence
[0103] The whole genome sequence of brewing yeast can be determined
mainly using the whole genome shotgun method. The DNA fragment of
which DNA sequence is determined can be prepared by a PCR using the
shotgun library prepared in the above (b). To be specific, clone of
the genome shotgun library is inoculated using a replicator
(manufactured by Gene Solution) to a 384-well titer plate where
about 50 .mu.l each of an ampicillin-containing LB medium is placed
to each well and cultured without shaking through the night at
about 30 to 37.degree. C. The culture is transferred using a
replicator (manufactured by Gene Solution) or the like to a
384-well reaction plate (manufactured by AB Gene) where about 10
.mu.l each of a reaction solution for PCR (TaKaRa Ex Taq
manufactured by Takara Shuzo) is placed, and PCR is carried out
according to a protocol by Makino, et al. (DNA Research, volume 5,
pages 1 to 9 (1998)) or the like using a GeneAmp PCR System 9700
(manufactured by Applied Biosystems) or the like, whereupon
amplification of the inserted fragment is carried out.
[0104] Excessive primer and nucleotide are removed using a kit for
the purification of PCR products (manufactured by Amersham
Bioscience), etc. and a sequence reaction is carried out using the
sample as a template.
[0105] Cosmid DNA from the cosmid library of (c) can be prepared by
the following method. That is, clone derived from cosmid library is
inoculated to each well of a 96-well plate where about 1.0 mL each
of an ampicillin-containing appropriate medium such as a 2.times.YT
medium (1.6% bactotryptone, 1% yeast extract and 0.5% sodium
chloride; pH 7.0) is placed and cultured with shaking through the
night at about 30 to 37.degree. C. Cosmid DNA from the said culture
can be prepared using KURABO PI-1100 AUTOMATIC DNA ISOLATION SYSTEM
(manufactured by KURABO) according to a manual of KURABO or the
like, and they can be used as templates for sequencing
reaction.
[0106] (e) Sequencing Reaction
[0107] A Sequencing reaction can be carried out using a
commercially available sequence kit, etc. Preferred examples of the
present invention are shown below.
[0108] A sequence reaction mixture can be prepared as follows. The
PCR product or cosmid DNA prepared in the above (d) is mixed with
about 2 .mu.l of DYEnamic ET Terminator Sequencing Kit
(manufactured by Amersham Bioscience) and appropriate primers to
give about 8 .mu.l of reaction mixture. An M13 forward (M13-21)
primer and an M13 reverse (M13RV) primer (manufactured by Takara
Bio), etc. are used for the sequence reaction of a PCR product
derived from shotgun DNA, while a forward primer such as SS-cos F.1
(SEQ ID NO: 7) and a reverse primer such as SS-cos R.1 (SEQ ID NO:
8), etc. are used for cosmid DNA. Amounts of the primer and the DNA
fragment are about 1 to 4 pmole and about 50 to 200 ng,
respectively.
[0109] A dye terminator sequence reaction of about 50 to 70 cycles
can be carried out using the reaction solution and GeneAmp PCR
System 9700 (manufactured by Applied Biosciences). When a
commercially available kit such as DYEnamic ET Terminator
Sequencing Kit is used, a cycle parameter follows a manual attached
thereto. Purification of the sample is carried out according to the
manual of Millipore using MultiScreen HV plate (manufactured by
Millipore), etc. The purified reaction product is precipitated with
ethanol and the resulting precipitate is dried and stored in a dark
place of about 4.degree. C. The dried product is analyzed using
commercially available sequencer and analyzer such as MegaBACE 1000
Sequencing System (manufactured by Amersham Bioscience) and ABI
PRISM 3700 DNA Analyzer (manufactured by Applied Biosystems), etc.
according to the manuals attached thereto.
[0110] (f) Reconstruction of Genomic Sequence by Means of Assembly
(A Process Whereby the Order of Multiple Sequenced DNA Fragments is
Determined)
[0111] Reconstruction of genomic DNA can be carried out from
sequence information of DNA fragments obtained in the above (4).
All operations of the reconstruction of genomic DNA sequence can be
carried out on an UNIX.RTM. platform. Base call can be conducted by
a software such as phred (The University of Washington) or the
like, masking of vector sequence can be carried out by a software
such as Cross Match (The University of Washington) or the like and
assembly can be carried out by a software such as Phrap (The
University of Washington) or the like. Contig obtained as a result
of assembly can be analyzed using a graphical editor such as
consed, a graphical editor (The University of Washington) or the
like. A series of works from base call to assembly can be carried
out en bloc utilizing phredPhrap, a script attached to the
consed.
[0112] (B) Comparison of the Whole Genome Sequence of Brewing Yeast
with that of S. cerevisiae
[0113] Comparison of the whole genome sequence obtained in (A) with
that of S. cerevisiae includes (g) Preparation of a comparative
database compiling the comparison data of each of DNA sequences of
both ends of cosmid and shotgun clone and contig with S. cerevisiae
genome sequence, and mapping of them on S. cerevisiae genome
sequence.
[0114] (g) Preparation of a Comparative Database Compiling the
Comparison Data of Each of DNA Sequences of Both Ends of Cosmid and
Shotgun Clone and a DNA Sequence of Contig with Genomic DNA
Sequence of S. cerevisiae, and Mapping of them on S. cerevisiae
Genome Sequence.
[0115] Widely used industrial yeast such as bottom fermenting yeast
(S. pastorianus) has been regarded as a natural hybrid of S.
cerevisiae and its closely related species (such as S. bayanus)
"Int. J. Syst. Bacteriol. volume 35, pages 508-511 (1985)". In view
of the above, DNA sequences of the both ends of cosmid clone
prepared in (e) are subjected to a homology searching against S.
cerevisiae genome sequence by a homology searching algorithm,
whereupon the homologous region and the identity of each DNA
sequence to S. cerevisiae genome sequence can be determined, thus
database can be prepared. An example of identity percentages
distribution graph of cosmid DNA sequence corresponding to S.
cerevisiae genome DNA sequence is shown in FIG. 2. The DNA sequence
of cosmid is roughly classified into a DNA sequence group showing
more than 94% identity to S. cerevisiae genome sequence and a DNA
sequence group showing around 84% identity thereto. Accordingly, a
DNA sequence showing more than 94% identity is named an Sc-type DNA
sequence derived from S. cerevisiae while a DNA sequence showing
around 84% identity is named a non-Sc-type DNA sequence derived
from a closely-related species of S. cerevisiae and, gene with the
Sc type DNA sequence or the non-Sc type DNA sequence is named Sc
type gene or non-Sc type gene, respectively.
[0116] Similarly, a comparative database of the DNA sequence of
both ends of shotgun clone prepared in (e) with genomic DNA
sequence of S. cerevisiae is prepared. On the basis of the
information obtained from the prepared comparative database, a
mapping of cosmid clone and shotgun clone on S. cerevisiae-genome
sequence is carried out (refer, for example, to FIG. 3). A
comparative database of the DNA sequence of the contig prepared in
(f) with S. cerevisiae genome sequence is also prepared and mapping
is carried out. Although the mapping technique is nearly the same
as that mentioned above, when contigs linked by paired
forward-reverse DNA sequence from the same cosmid and shotgun
clone, those contigs are linked (refer, for example, to FIG.
4).
[0117] (C) Selection of the Gene of Bottom Fermentation Yeast
Encoding an Amino Acid Sequence Having 70 to 97% Identity to an
Amino Acid Sequence Encoded by the Gene of S. cerevisiae
[0118] A stage for the selection of the gene of bottom fermenting
yeast encoding an amino acid sequence having 70 to 97% identity to
an amino acid sequence encoded by the gene of S. cerevisiae
includes (h) a process of identification of ORF (open reading
frame) and assignment of function.
[0119] (h) Identification of ORF and Assignment of Its Function
[0120] Identification of ORF in the DNA sequence assembled in (f)
is carried out. Preferred examples are specifically mentioned
below. With regard to a certain length DNA sequence (such as not
less than 150 base) embraced by initiation codon and termination
codon, there can be carried out identification of ORF existing in a
DNA sequence assembled in (f) using a program, such as ORF finder
(http://www.ncbi.nih.gov/gorf/gorf.ht- ml) or the like for the
identification of ORF for six kinds of reading frames including
complementary sequence.
[0121] Assignment of function of protein encoded by the identified
ORF can be carried out using a homology searching such as BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/) or the like to an amino acid
sequence of ORF of S. cerevisiae that has been registered and
published in the Saccharomyces Genome Database (SGD:
http://genome-www.stanford.edu- /Saccharomyces/).
[0122] On the other hand, it is possible to analyze the chromosomal
structure of a brewing yeast by DNA microarray-based comparative
genomic hybridization and PCR.
[0123] Yeast genomic DNA is prepared using a Quiagen Genomic Tip
100/G (#10243) and Qiagen Genomic DNA Buffer Set (#19060) according
to the manual attached to the kit. The DNA (10 .mu.g) is digested
with DNase I (manufactured by Invitrogen) according to a method of
Winzeler, et al. (Science, volume 281, pages 1194-1197 (1998)),
biotinylated using a terminal transferase (manufactured by Roche)
and hybridized to a DNA microarray (Affymetrix Gene Chip Yeast
Genome S98 Array). Hybridization and detection of the signal
intensity of microarray are carried out using a Gene Chip Analysis
Basic System and analysis soft ware (Microarray Suite 5.0)
manufactured by Affymetrix.
[0124] The signal of each probe hybridized with the DNA of brewing
yeast is normalized to that of the haploid laboratory yeast strain
S288C using an analysis soft ware (Microarray Suite 5.0) and shown
as signal log ratio (2.sup.n). Signal log ratios were lined
following genes order in each chromosome using a spreadsheet
program (Microsoft Excel 2000) and the signal log ratios are shown
in bar graphs (refer, for example, to FIG. 5). The non-Sc type
genes do not hybridize to the S. cerevisiae array, therefore, the
Sc type gene dosage affect the signal log ratio and the points
where the signal log ratios show vigorous changes are considered to
be translocation sites between Sc type and non-Sc type
chromosome.
[0125] The chimera chromosome structure can be confirmed by PCR
where a genomic DNA derived from brewing yeast is used as a
template and Sc type and non-Sc type shotgun sequences is used as
primers.
[0126] PCR is carried out using a Takara PCR Thermal Cycler SP
according to the attached manual using a Takara LA Taq.TM. and a
buffer attached thereto.
[0127] As a result of the PCR, it is confirmed by 0.8% agarose
electrophoresis that a certain length of DNA fragment is amplified
from the brewing yeast. When a genomic DNA of S. cerevisiae which
is a laboratory strain is used as a template for the PCR, no
amplification of DNA fragment is detected. If DNA sequences of the
both ends of the DNA fragment amplified from the brewing yeast are
further confirmed, it is consistent with the genome sequences
determined by a shotgun method and it can be confirmed that, within
such region, translocation between Sc type and non-Sc type
chromosome takes place, whereupon a chimera chromosome is
formed.
[0128] (D) Functional Analysis of Genes Derived from the Bottom
Fermenting Yeast
[0129] The stage of functional analysis of gene includes (i)
selection of the gene, (i') cloning of the gene, (j) functional
analysis of the gene by disruption and (k) functional analysis of
the gene by overexpression.
[0130] (i) Selecting of the Gene
[0131] There is no particular limitation for the methods used for
the selection of gene(s) for functional analysis, while preferred
methods are, for example, a method using the assignment of the
function obtained in the above (h) and a method using a DNA
microarray as described below. The method using DNA microarray is,
for example, gene expression analysis to identify genes, which show
characteristic expression profile under some conditions, or
comparative genomic hybridization to identify genes, which have
different copy numbers or different DNA sequences, by detecting
deference of signal intensities of probes.
[0132] (i') Cloning of the Gene
[0133] Genes selected in the above (i) can be obtained from the
bottom fermentaing yeast according to a common method mentioned,
for example, in Molecular Cloning, Third Edition. That is,
oligonucleotides having sequences adjacent to the gene are
synthesized and a common PCR cloning method is carried out using a
genomic DNA prepared from a bottom fermenting yeast as a template,
whereupon the selected gene can be isolated and obtained. With
regard to DNA sequences obtained as such, for example, by SEQ ID
NO: 1 or NO: 2 may be listed.
[0134] When the gene is named, for example, a gene{circle over
(1)}, the gene{circle over (1)} or primer for amplifying the
gene{circle over (1)} by a PCR method may be also synthesized using
a polynucleotide synthesizer on the basis of the above-mentioned
sequence information. In addition, gene{circle over (1)} means not
only a DNA fragment having the same DNA sequence as gene {circle
over (1)} but also a DNA fragment hybridizing to the above gene
under stringent condition. The DNA fragment which hybridizes under
stringent condition means a DNA fragment which is obtained by a
colony hybridization method, a plaque hybridization method, a
southern blot hybridization method or the like, using the DNA
fragment containing the sequence of the gene {circle over (1)}
identified in the above as a probe. To be specific, a DNA fragment
which shows at least not less than 60% identity to a DNA sequence
of the gene {circle over (1)}, preferably not less than 80%
identity thereto and, more preferably, not less than 95% identity
thereto may be listed. The hybridization may be carried out
according to a method mentioned in "Molecular Cloning, Third
Edition", "Current Protocols in Molecular Biology, John Wiley &
Sons (1987-1997) (hereinafter, abbreviated as Current Protocols in
Molecular Biology), "DNA Cloning 1: Core Techniques, A Practical
Approach, Second Edition, Oxford University (1995)", and the
like.
[0135] To be more specific, shotgun clone containing full-length of
the above-mentioned gene {circle over (1)} can be retrieved using
the comparative database obtained in (g) and, on the basis of
homology and positional information, etc. When there is no clone
containing full-length of the gene in the shotgun library, a DNA
fragment encoding the full length of the gene is prepared by a PCR
method. For example, a DNA fragment containing the above-mentioned
gene is obtained using synthetic DNA primer pair represented by SEQ
ID NO: 13 and SEQ ID NO: 14, etc. Similarly, PCR is carried out
using a primer pair designed on the basis of the published
information of SGD and using genomic DNA of S. cerevisiae or bottom
fermenting yeast as a template, whereupon the full length of the Sc
type gene corresponding to the non-Sc type gene is prepared. For
example, using synthetic oligonucleotides of SEQ ID NO: 15 and NO:
16 as the primer pair, the DNA fragment containing the Sc type gene
can be obtained.
[0136] Sc or non-Sc type DNA fragment prepared as mentioned above
is inserted into, for example, pCR 2.1-TOPO vector attached to a TA
cloning kit (Invitrogen) using a TA cloning kit or the like,
whereupon a recombinant vector TOPO/Sc gene and TOPO/non-Sc gene
containing the DNA fragment having the Sc and the non-Sc type gene,
respectively, are able to be prepared. DNA sequences of the Sc and
non-Sc type DNA fragments can be comfirmed by a Sanger's method "F.
Sanger, Science, volume 214, page 1215, 1981".
[0137] (j) Functional Analysis of the Gene by Disruption
[0138] According to a method of the document "Goldstein, et al.,
Yeast, volume 15, page 1541, (1999)", it is possible to prepare a
DNA fragment for gene disruption by PCR where a plasmid containing
a drug-resistance gene (such as pFA 6a (G418r), pAG 25 (nat1)) is
used as a template. As a primer pair for the PCR, non-ScSSU1_for
(SEQ ID NO: 17)/non-ScSSU1_rv (SEQ ID NO: 18) or the like is used
for the non-ScSSU1 disruption, while for the Sc SSU1 disruprion,
ScSSU1_for (SEQ ID NO: 19)/ScSSU1_rv (SEQ ID NO: 20) or the like is
used. For the non-Sc type gene disruption, it is also possible to
use a plasmid such as pPGAPAUR (AUR1-C) and a primer pair such as
non-ScSSU1_for +pGAPAUR (SEQ ID NO: 21)/non-ScSSU1_rv+AURI-C (SEQ
ID NO: 22).
[0139] A bottom fermenting yeast is transformed with the DNA
fragment for the gene disruption prepared by the above-mentioned
method. The transformation may follow a method mentioned in the
Japanese Patent Laid-Open Gazette No. 07/303,475. Further, the
concentration of the drug for the selection of transformants may be
appropriately determined by investigating the sensitivity of the
yeast used as a host.
[0140] With regard to the transformant prepared here, it is
comfirmed that each of the drug-resistance genes is introduced and
the said gene is disrupted correctly using a Southern analysis. To
be specific, the genomic DNA extracted from the parental strain and
the transformant are firstly digested by an appropriate restriction
enzyme to distinguish Sc and non-Sc type gene (for example, at
37.degree. C. for 18 hours), then fractionated with 1.5% agarose
gel electrophoresis and transferred to a membrane. After that, they
are hybridized to a probe specific to an Sc-type or a non-Sc type
gene for example at 55.degree. C. for 18 hours according to a
protocol of Alkphos Direct Labelling Reagents (Amersham) and a
signal is detected by CDP-Star.
[0141] The function of the gene obtained in (i') can be confirmed
by fermentation test using a parental strain and SSU1 disruptants
prepared in the above (j) and comparison of their fermentation
character. Fermentation test can be carried out, for example, using
wort under the following condition.
[0142] Original extract: about 10 to 15%
[0143] Fermentation scale: 1 to 3 liters
[0144] Dissolved oxygen concentration: about 8 to 10 ppm
[0145] Fermentation temperature: about 15.degree. C.
[0146] Pitching rate: about 4 to 6 g of wet yeast cells/L
[0147] Wort is periodically sampled and monitored in cell growth
(OD 600), apparent extract, the concentration of the substance
participating in the function of the gene obtained in (i'), etc. is
analyzed. For example, when the function of the gene obtained in
(i') participates in discharge of sulfite, the sulfite
concentration in the wort during the fermentation is analyzed.
Quantitative analysis of sulfite is carried out in such a manner
that sulfite is captured in a hydrogen peroxide solution by means
of distillation under an acidic condition and subjected to
titration with an alkali (Revised Method for BCOJ Beer Analysis by
the Brewing Society of Japan).
[0148] (k) Functional Analysis of the Gene by Overexpression
[0149] A DNA fragment containing the full-length of the non-Sc type
gene is excised by an appropriate restriction enzyme from the
plasmid TOPO/non-Sc gene prepared in (i'). It is inserted into a
cloning site of a vector for gene expression such as pNI-NUT to
construct a vector (pYI-non-Sc type gene) for overexpression of the
non-Sc type gene. The vector pNI-NUT contains URA3 as a homologous
recombination site and nourseothricin-resistance gene (nat1) and
ampicillin-resistance gene (Amp.sup.r) as selective markers. On the
other hand, a vector for overexpression of the Sc type gene (pNI-Sc
type gene) has a structure where the above-mentioned pYI-non-Sc
type gene is substituted by the corresponding Sc type gene. For
overexpression of the Sc or non-Sc type gene introduced here,it is
preffered to be driven by promoter and terminator of constitutively
expressing gene, for example, glyceraldehyde-3-phosphate
dehydrogenase gene (TDH3).
[0150] A bottom fermenting yeast is transformed using the
overexpression vector, which is prepared by the above-mentioned
method. The transformation is carried out by the method mentioned
in the Japanese Patent Laid-Open Gazette No. 07/303,475 and
transformants are selected on an appropriate selective medium.
Confirmation of the overexpression may be carried out by RT-PCR
method, etc. Extraction of the total RNA may be carried out using
an RNeasy Mini Kit (Qiagen) or the like, according to the manual of
"for total RNA isolation from yeast" attached to the kit. For
example, it is possible to use ScSSU1_for331 (SEQ ID NO:
23)/ScSSU1.sub.--982rv (SEQ ID NO: 24) and nonSc-SSU1_for329 (SEQ
ID NO: 25)/nonSc-SSU1.sub.--981rv (SEQ ID NO: 26) as specific
primer pairs for the amplification of Sc and non-ScSSU1 gene,
respectively. To amplify the constitutively expressed gene, for
example PDA1, as an internal standard, PDA1_for1 (SEQ ID NO:
27)/PDA1.sub.--730rv (SEQ ID NO: 28) etc. may be used as a specific
primer pair. PCR product is fractionated with 1.2% agarose gel
electrophoresis and detected with ethidium bromide staining. The
overexpression of the said gene in the transformant is confirmed by
comparison of quantity of the PCR products.
[0151] The functional analysis of the gene obtained in (i') can be
carried out by a fermentation test using the parental strain and
the overexpressed strain prepared in the above (k). Fermentation
test may be carried out under the condition mentioned in (j).
[0152] According to the same method mentioned in (j), the wort is
periodically sampled and monitored in the cell growth (OD600),
apparent extract and the concentration of the substance
participating in the function of the gene obtained in (i').
[0153] With regard to the DNA which is obtained by the screening
method of the present invention, a DNA containing the DNA sequence
of the non-Sc type gene obtained in the above and a DNA which
hybridizes to the said DNA under stringent condition may be
listed.
[0154] The DNA obtained by the screening method of the present
invention includes single-stranded and double-stranded DNAs
although they are non-limitative. A DNA which hybridizes to the DNA
containing a DNA sequence of the non-Sc type gene obtained in the
above under stringent condition includes a degenerated mutant of
codon of the protein encoded by the said gene. A degenerated mutant
means a polynucleotide fragment encoding the same amino acid
sequence by degeneration of codon, although in terms of a DNA
sequence, it is different from a DNA sequence of the non-Sc type
selected by the present invention.
[0155] Specific examples thereof are a DNA with a sequence as shown
by SEQ ID NO: 1 or 2, a DNA which hybridizes to the said DNA under
stringent condition, etc. The DNA which hybridizes under stringent
condition means a DNA which is prepared by a colony hybridization
method, a plaque hybridization method, a southern blot
hybridization method or the like using a DNA fragment with the
sequence of the non-Sc type identified hereinabove as a probe.
[0156] Hybridization may be carried out according to a method
mentioned in "Molecular Cloning, Third Edition", "Current Protocols
in Molecular Biology", "DNA Cloning 1: Core Techniques, A Practical
Approach, Second Edition, Oxford University (1995)", etc. Specific
examples of the hybridizable DNA is a DNA which shows at least not
less than 60% identity, preferably a DNA which shows not less than
80% identity and, more preferably, a DNA which shows not less than
95% identity to a DNA sequence as shown in SEQ ID NO: 1 or 2 when
calculation is conducted using a parameter of the default setting
(initial setting) by a software for homology searching such as
FASTA, BLAST, Smith-Waterman "Meth. Enzym., volume 164, page 765
(1988)", etc.
[0157] An example of the DNA obtained by the screening method of
the present invention is a DNA encoding a polypeptide comprising an
amino acid sequence as shown by SEQ ID NO: 3 or 4 or a DNA which
hybridizes to the said DNA under stringent condition.
[0158] An example of the polypeptide which is encoded by the DNA
obtained by the screening method of the present invention is a
polypeptide encoded by the DNA containing the DNA sequence of ORF
obtained in the above and a polypeptide encoded by the DNA which is
hybridized to the said DNA under stringent condition or a
polypeptide comprising an amino acid sequence as shown by SEQ ID
NO: 3 or 4.
[0159] Further, a polypeptide comprising an amino acid sequence
where one or more amino acid residue(s) is/are deficient and/or
substituted and/or added in the amino acid sequence of the said
polypeptide and has substantially same activity as the activity of
the said polypeptide is also included in the present invention. The
expression reading "substantially same activity as the activity of
the said polypeptide" means the same activity as the activity which
is represented by enzymatic activity or the function inherent to
the polypeptide before the deficiency, substitution or addition.
The said polypeptide can be prepared by a site-specific mutation
introduction which is mentioned in "Molecular Cloning, Third
Edition", "Current Protocols in Molecular Biology", "Nuc. Acids.
Res., volume 10, page 6487 (1982)", "Proc. Natl. Acad. Sic. USA,
volume 79, page 6409 (1982)", "Gene, volume 34, page 315 (1985)",
"Nuc. Acids. Res., volume 13, page 4431 (1985)", "Proc. Natl. Acad.
Sci. USA, volume 82, page 488 (1985)", etc. For example, it is able
to be prepared by introducing a site-specific mutation into a DNA
encoding a polypeptide comprising an amino acid sequence as shown
in SEQ ID NO: 3 or 4. Although there is no particular limitation
for the number of the amino acid residue(s) which is/are deficient
and/or substituted and/or added, the number is within such an
extent that is able to be deficient and/or substituted and/or added
by known methods such as the above-mentioned site-specific mutation
method and is one to several tens, preferably 1 to 20, more
preferably 1 to 10 and, still more preferably, 1 to 5.
[0160] The DNA of one or more amino acid residue(s) is/are
deficient and/or substituted and/or added in the amino acid
sequence of the polypeptide of the present invention means that
there is/are one or more deficiency (ies) and/or substitution(s)
and/or addition(s) of one or more amino acid residue(s) in any one
or more position(s) of the amino acid sequence in the same
sequence. Those deficiency (ies) and/or substitution(s) and/or
addition(s) may take place at the same time and the substituted or
added amino acid residue may be either naturally occurring type or
a non-naturally occurring type. Examples of the amino acid residue
of a natural type are L-alanine, L-asparagine, L-aspartic acid,
L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine,
L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and
L-cysteine, etc.
[0161] Examples of the amino acid residue which is able to be
substituted each other will be shown below. Amino acid residues in
the same group may be substituted each other.
[0162] Group A: leucine, isoleucine, norleucine, valine, norvaline,
alanine, 2-aminobutanoic acid, methionine, O-methylserine,
tert-butylglycine, tert-butylalanine and cyclohexylalanine.
[0163] Group B: aspartic acid, glutamic acid, isoaspartic acid,
isoglutamic acid, 2-aminoadipic acid and 2-aminosuberic acid.
[0164] Group C: asparagine and glutamine.
[0165] Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic
acid and 2,3-diaminopropionic acid.
[0166] Group E: proline, 3-hydroxyproline and 4-hydroxyproline.
[0167] Group F: serine, threonine and homoserine.
[0168] Group G: phenyl alanine and tyrosine.
[0169] For the purpose that the resulting mutated polypeptide has
the substantially same activity as the activity of the polypeptide
before the mutation, it is preferred that the mutated one has at
least 60% or more, usually 80% or more or, particularly, 95% or
more of identity to the amino acid sequence of the polypeptide
before the mutation when calculation is carried out using a
parameter of the default setting (initial setting) by a software
for the analysis such as BLAST and FASTA.
[0170] It is also possible to produce the polypeptide of the
present invention by a chemical synthetic method such as Fmoc
method (fluorenylmethyloxycarbonyl method), tBoc method
(tert-butyloxycarbonyl method), etc. It is further possible to
chemically synthesize by using a peptide synthesizers manufactured
by Advanced ChemTech, Perkin-Elmer, Pharmacia, Protein Technology
Instrument, Synthecell-Vega, PerSeptive, Shimadzu, etc.
[0171] When the method of the present invention is used, it is
possible to determine the whole genome sequence of industrial
yeast, to identify the useful genes of industrial yeast and to
assign the functions of the said genes. There are many cases where
the genes in industrial yeast are industrially useful and, when the
genes are classified on the basis of the assigned functions,
character of the yeast is clarified and precious information for
breeding of industrial yeast is able to be obtained. For example,
when the industrial yeast is a brewing yeast, then a gene
participating in increase in productivity and improvement in flavor
in the production of alcoholic beverage is identified and, in case
the gene is disadvantageous for the increase of productivity or for
the improvement of flavor, the gene expression is suppressed by a
gene disruption, an antisense method or an RNAi method (c.f., for
example, Non-Patent Document 10), whereupon yeast which shows an
excellent brewing character can be bred. In case the gene is
advantageous for the increase of productivity, improvement of
flavor, etc., then for example the gene is overexpressed in the
yeast, whereupon brewing yeast which shows an excellent brewing
character, which is industrially useful, can be bred.
[0172] An example where the gene obtained by the screening method
of the present invention is used to breed useful yeast is shown as
follows.
[0173] As already mentioned above, when the sulfite concentration
in a product is increased, it is possible to make a product with
excellent flavor stability. Therefore, if the gene obtained by the
screening method of the present invention contributes to production
and efflux of sulfite, it is now possible that a transformant is
cultivated and expressed the said gene to make a product with
excellent flavor stability as a result of the increase in the
concentration of sulfite in the product.
[0174] It has been known that a bottom fermenting yeast reduces
sulfate ion (SO.sub.4.sup.2-) taken from outside of the cell to
sulfite ion (SO.sub.3.sup.2-). However, sulfite inhibits
glyceroaldehyde-3-phosphate dehydrogenase and reduces the
concentration of intracellular ATP, therefore, yeast has a function
of discharging sulfite so that excessive sulfite should not be
accumulated in the cell. SSU1 is a gene, which has been isolated
and shown complement the sulfite-sensitive mutation (c.f., for
example, Non-Patent Document 11). SSU1 gene product comprises 485
amino acid residues, and the structural analysis suggests that it
is a transporter with 9 to 10 membrane-spanning domains (c.f., for
example, Non-Patent Document 12). Further, as a result of
experiment using a SSU1 overexpressed strain, it has been already
proved that the SSU1 gene product participates in discharge of
sulfite (c.f., for example, Non-Patent Document 13).
[0175] Bottom fermenting yeast usually has a high production
ability of sulfite, while top fermenting yeast rarely produces it.
By using a screening method of the present invention, it is
possible to select non-ScSSU1 gene which is specific to bottom
fermenting yeast in addition to ScSSU1 gene which exists in both
top and bottom fermenting yeast. Similarly, in the case of MET14
gene, which encodes a protein participating in the production of
sulfite, it is also possible to select a non-ScMET14 which is
specific to bottom fermenting yeast. Functions of, for example,
non-ScSSU1 and non-ScMET14 greatly participate in a high production
ability of sulfite, which is specific to bottom fermenting yeast,
and it is effective to intensify those non-ScSSU1 gene,
non-ScMET14, etc. in order to breed yeast which shows higher
production ability of sulfite.
[0176] Breeding methods of yeast where those non-ScSSU1 gene and
non-ScMET14 are intensified are specifically mentioned in the
EXAMPLES
[0177] With regard to yeast used as a host in the introduction of
gene selected by the screening method of the present invention,
there is no particular limitation so far as it is yeast which is
usable for brewing, and any yeast which is widely used as a brewing
yeast at present such as beer yeast including BH 84, NBRC 1951,
NBRC 1952, NBRC 1953 and NBRC 1954 may be used. Further, whisky
yeasts (such as S. cerevisiae NCYC 90), wine yeasts (such as wine
yeast Kyokai No. 1, No. 3, No. 4, etc.) and sake yeasts (such as
sake yeast Kyokai No. 7, No. 9, etc.) may be also used.
[0178] With regard to a vector used for the introduction of gene
into the above-mentioned host, there is no particular limitation so
far as it is a vector which can express gene in the yeast, and any
of plasmid of a multicopy (YEp type), a single-copy plasmid (YCp
type) and a chromosomal DNA-integrating plasmid (YIp type) may be
utilized. An example of a YEp vector is YEp 51 (J. R. Broach, et
al., Experimental Manipulation of Gene Expression, Academic Press,
New York, 83, 1983), etc. an example of a YCp vector is YCp 50 (M.
D. Rose, et al., Gene, volume 60, page 237, 1987), etc.; and an
example of a YIp vector is YIp 5 (K. Struhl, et al., Proc. Natl.
Acad. Sci. USA, volume 76, page 1035, 1979), etc. Those plasmids
are put into the market and are easily available.
[0179] The above-mentioned vector may have other sequence for
controlling expression of gene in yeast such as, promoter,
operator, enhancer, silencer, ribosome binding sequence,
terminator, etc. With regard to a promoter and a terminator for a
constitutive expression of a gene, there is no particular
limitation but any combination may be used so far as it functions
in a brewing yeast and is independent from sulfite concentration in
the product. As to a promoter for example, it is possible to use a
promoter for glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene,
a promoter for phosphoglycerate kinase (PGK1) gene, etc. Those
promoters have been known, and PGK1 gene, for example, is mentioned
in detail in publicly known documents such as M. F. Tuite, et al.,
EMBO J., volume 1, page 603 (1982) and is easily available.
[0180] It is not necessary that the above-mentioned other sequences
which regulate the expression of the introduced gene are
particularly provided from vector so far as the DNA obtained by the
screening method of the present invention includes them. When such
other sequences are not contained in the said DNA, it is preferred
that other sequences are prepared separately and ligated to the
said DNA. Alternatively, even in the case of higher expression
level or specific regulation of expression is required, other
sequences appropriate for such a purpose are ligated to the said
DNA.
[0181] A method for the transformation of the above vector to a
host may follow known procedures. For example, the following
methods may be used; an electroporation method "Meth. Enzym.,
volume 194, page 182 (1990)", a spheroplast method "Proc. Natl.
Acad. Sci. USA, volume-75, page 1929 (1978)", a lithium acetate
method "J. Bacteriology, volume 153, page 163 (1983)", a method
mentioned in "Proc. Natl. Acad. Sci. USA, volume 75, page 1929
(1978)", etc.
[0182] To be more specific, a host is cultivated in a standard
yeast nutrient medium (such as YEPD medium "Genetic Engineering,
vol. 1, Plenum Press, New York, 117 (1979) ", etc.) so that the
absorbance at 600 nm becomes 1 to 6. Cells are collected by
centrifugation, washed and subjected to a pre-treatment with an
alkali metal ion or, preferably, lithium ion in a concentration of
about 1M to 2M. After the cells are incubated at about 30.degree.
C. for about 60 minutes, they are incubated together with DNA to be
introduced (about 1 to 20 .mu.g) at about 30.degree. C. for about
60 minutes. Polyethyleneglycol or, preferably, polyethyleneglycol
of about 4,000 daltons is added as the final concentration will be
about 20% to 50%. After the incubation is carried out at about
30.degree. C. for about 30 minutes, the cells are subjected to a
heating treatment at about 42.degree. C. for about 5 minutes.
Preferably, the cell suspension is washed with a standard yeast
nutrient medium and placed in a predetermined amount of a fresh
standard yeast nutrient medium, then incubated at about 30.degree.
C. for about 1 hour. After the incubation, it is spread on an
appropriate selective medium plate.
[0183] Besides the above, as for a general cloning technique,
"Molecular Cloning, Third Edition", "Methods in Yeast Genetics, A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.)", etc. were referred to.
[0184] With regard to a selective marker used for the
transformation, it is not possible to utilize an auxotrophic marker
in the case of brewing yeast and, therefore, G 418-resistance gene
(G 418.sup.r), copper-resistance gene (CUP 1) "M. Marin, et al.,
Proc. Natl. Acad. Sci. USA, volume 81, page 337, 1984",
serulenin-resistance gene (fas2m, PDR 4) ("Atsushi Inogoshi, et
al., Seikagaku, volume 64, page 660, 1992", "M. Hussain, et al.,
Gene, volume 101, page 149, 1991", etc. are applicable.
[0185] The brewing yeast bred according to the present invention is
not different from a parental strain in terms of growth and
fermentation ability of yeast. Accordingly, materials, facilities
for the production, production control, etc. may be entirely the
same as those in the conventional methods, which is an important
aspect of the present invention. However, it goes without saying
that, conditions such as fermentation period may be changed on a
case-by-case, if desired. For example, when a brewing yeast in
which discharging ability of sulfite is intensified and an
alcoholic beverage is produced using such yeast, only the content
of sulfite in the product changes, and there is no difference from
the case where a parental strain is used, in terms of growth and
fermentation ability of the yeast. Accordingly, materials,
facilities for the production, production control, etc. may be
entirely the same as those in the conventional methods, and there
is no increase in the cost of production of an alcoholic beverage
in which sulfite content increases and of which flavor is
improved.
[0186] (E) Production of a DNA Array of this Invention
[0187] A DNA array of this invention can be produced based on the
DNA sequence information of the ORFs obtained in the above (f).
Examples include a DNA array comprising a solid support to which at
least one of a polynucleotide comprising the DNA sequence obtained
above items (f), a polynucleotide which hybridizes with the
polynucleotide under stringent conditions, and a polynucleotide
comprising 10 to 200 continuous nucleotides in the DNA sequence of
the polynucleotide is adhered; and a DNA array comprising a solid
support to which at least one of a polynucleotide encoding a
polypeptide comprising the amino acid sequence obtained as above
(h), a polynucleotide which hybridizes with the polynucleotide
under stringent conditions, and a polynucleotide comprising 10-200
continuous bases in the DNA sequence of the polynucleotides, a
polynucleotide comprising intergenic DNA sequence between two ORFs
deduced from the above (h) is adhered.
[0188] DNA arrays of the present invention include substrates known
in the art, such as a DNA chip, polynucleotide array and a DNA
microarray and a DNA macroarray, or the like, and comprises a solid
support and plural polynucleotides of fragments thereof which are
adhered to the surface of the solid support. As the polynucleotids
or oligonucleotides adhered to the solid support, the
polynucleotides or oligonucleotides of the present invention
obtained in the above items (f) and (h) can be used. The analysis
described below can be efficiently performed by adhering the
polynucleotides or oligonucleotids to the solid support at a high
density, though a high fixation density is not always necessary.
Apparatus for achieving a high density, such as an arrayer robot or
the like, is commercially available from Takara Shuzo (GMS417
Arrayer), and the commercially available product can be used. Also,
the oligonucleotide of the present invention can be synthesized
directly on the solid support by the photolithography method or the
like (Nat. Genet. 21, 20-24 (1999)). In this method, a linker
having a protective group which can be removed by light irradiation
is first adhered to a solid support, such as slide glass or the
like. Then, it is irradiated with light through a mask (a
photolithograph mask) permeating light exclusively at a definite
part of the adhesion part. Next, an oligonucleotide having a
protective group which can be removed by light irradiation is added
to the part. Thus, a ligation reaction with the nucleotide arises
exclusively at the irradiated part. By repeating this procedure,
oligonucleotides, each having a desired sequence, different from
each other can be synthesized in respective parts. Usually, the
oligonucleotides to be synthesized have a length of 10 to 30
nucleotides. There is no particular limitation for the methods used
for the production of DNA array and the method may be conducted
according to the known means, while preferred method for each of
them is mentioned below.
[0189] (l) Production of a DNA Array
[0190] (l)-1 Solid Support
[0191] Any materials of which the polynucleotids or fragments can
be adhere to the surface can be used as the solid supports for the
invention DNA array. There is no particular limitation for the
material and shape used for the solid support, while preferred
materials are some resinoids, such as polycarbonate, plastics or
the like, as a material and a plate-like and film-like as a
solid.
[0192] (l)-2 Selection a Oligonucleotide
[0193] The example of oligonucleotides to be fixed on the plate of
a DNA array of this invention are as follows. Based on the DNA
sequences of ORFs obtained in the above (h) and/or intergenic DNA
seqqeunces deduced from the above (h), unique and complementary
probes (PM Probe; Perfect Match Probe) against whole genome
sequence of brewing yeast can be designed using a certain method of
probe production, such as GeneChip.RTM. (Affymetrix) technology or
the like. Examples of these probes are (i) an oligonucleotide
having 10 to 30 nucleotides existing in an open reading frame of
the whole genome sequence of an industrial yeast and not existing
in the region other than the region of said 10 to 30 nucleotides
sequence in the whole genome sequence, (ii) an oligonucleotide
having an DNA sequence complementary to the DNA sequence of
oligonucleotide described in (i), (iii) an oligonucleotide
hybridizing in a stringent condition to the oligonucleotides
described in (i) and (ii). The other examples of these probes are
(iv) an oligonucleotide having 10 to 30 nucleotides existing in a
non-coding region of the whole genome sequence of an industrial
yeast and not existing in the region other than the region of said
10 to 30 nucleotides sequence in the whole genome sequence, (v) an
oligonucleotide having an DNA sequence complementary to the DNA
sequence of oligonucleotide described in (iv), (vi) an
oligonucleotide hybridizing in a stringent condition to the
oligonucleotides described in (iv) and (v). Nucleotides number of
these oligonucleotides are not limited, but 10 to 30 nucleotides
are preferable. 11-50 probes an each locus can be designed with
focus on 3' prime side of each locus, as the use of sets of probes
for each locus can provide redundancy in the detection and analysis
of the data, can mitigate the potentially confounding effects of
occasional cross-hybridization, and can make it so all probes do
not have to hybridize identically in order to obtain quantitative
information. To further increase the sensitivity and specificity of
detection, each PM probe can be designed with a closely related
mismatch probe (MM probe) that is identical to PM probe with the
exception of a mismatched base, i.e. base 13. The preferable length
of oligonucleotide which is used in this invention is 26 base, but
no particular limitation for the length of oligonucleotide.
[0194] (l)-3 Adhering Oligonucleotides to Solid Support
[0195] There is no particular limitation for the methods used for
adhering oligonucleotides to solid support, and the method may be
conducted according to the known means, while preferred method is
mentioned below. For example, all of designed PM and MM probes as
above ((l)-2) can be adhered to the surface of solid support to
produce a DNA array using a certain method, such as GeneChip.RTM.
technology or the like.
[0196] There is no particular limitation for the methods used for
analysis using DNA maicroarray, while preferred methods for each of
them is mentioned below, i.e., the example of gene expression
analysis to identify genes, which show characteristic expression
profile under some conditions, classification of industrial yeast,
detection of nucleotide polymorphism and selection of genes for
functional analysis are mentioned below.
[0197] (m) Gene Expression Analysis
[0198] Gene expression analysis of brewing yeast can be carried out
using the DNA array of this invention produced according to the
method described in (l). It is possible to identify the highly
inducible or reducible gene(s) according to change of not only
medium but also environment using the DNA array. It is also
possible to identify the specific gene(s) for lager brewing yeast
in brewing using the DNA array. But it is not limited for these
examples.
[0199] Gene expression analysis includes culturing of a industrial
yeast, preparation of mRNA, synthesis of labeled cRNA (or cDNA),
hybridization, and data analysis. There is no particular limitation
for the methods of gene expression analysis, while preferred
methods for each of them is mentioned below.
[0200] (m)-1 Culturing a Industrial Yeast in a Various
Condition
[0201] Industrial yeast can be cultivated under various conditions
for any purpose. For example, the cultivation for identification of
genes which respond to the change of composition of culture medium
can be carried out as mentioned below. Industrial yeast can be
grown overnight in a Zinc replete medium, such as LZMM medium+40
.mu.M zinc sulfate at 30.degree. C. with shaking. LZMM medium
contains 0.17% yeast nitrogen base w/o amino acids (manufactured by
DIFCO), 0.5% ammonium sulfate, 20 mM sodium citrate (pH 4.2), 125
.mu.M MnCl2, 10 .mu.M FeCl2, 2% maltose, 10 mM EDTA (pH 8.0), or
the like. Cells are harvested and washed three times with sterile
distilled water. An adequate amount of cells, are inoculated to an
optical density (OD600) of 0.25, or the like, in 1) zinc depleted
medium (LZMM medium) or the like, 2) zinc replete medium (LZMM+40
.mu.M zinc sulfate) or the like, 3) oxidative stress medium
(LZMM+40 .mu.M zinc sulfate+2 mM H.sub.2O.sub.2) or the like, 4)
carbon starvation medium (deleting maltose from above LZMM+40 .mu.M
zinc sulfate) or the like. Cells are grown at 30.degree. C. for 6
hours or the like and harvested for RNA preparation. Cells
withdrawn from fermentation tube under beer fermenting condition
can be used for the following experiments.
[0202] (m)-2 Preparation of mRNA
[0203] Preparations of total RNA can be carried out using an
RNeasy.RTM. Mini Kit (manufactured by QIAGEN) or the like according
to a manual. Preparations of Poly (A)+ mRNA from total RNA are
carried out using an Oligotex Direct mRNA kit (manufactured by
QIAGEN) or the like according to a manual. There is no particular
limitation for the methods used for preparation of mRNA and the
method may be conducted according to the known means.
[0204] (m)-3 Synthesis of Labeled cRNA
[0205] Synthesis of Labeled cRNA can be carried out using a
BioArray HighYield RNA Transcript Labeling Kit (manufactured by
Affymetrix) or the like according to a manual. Biotin can be used
for labeling. There is no particular limitation for the methods
used for syntheses of Labeled cRNA and the method may be conducted
according to the known means.
[0206] (m)-4 Hybridization
[0207] 5 .mu.g of Biotin-Labeled cRNA, 1.711 of 3 nM Control
Oligonucleotide B2 (manufactured by Affymetrix), 5 .mu.l of
20.times.Eukaryotic Hybridization Controls (manufactured by
Affymetrix), 1 .mu.l of 10 mg/ml Herring Sperm DNA (manufactured by
Affymetrix), 1 .mu.l of 50 mg/ml Acetylated BSA (manufactured by
Affymetrix), 50 .mu.l of 2.times. Hybridization buffer
(manufactured by Affymetrix), and water (manufactured by
Affymetrix) to give final volume of 100 .mu.l are mixed and
hybridized to the DNA array according to a Technical Mannual of
Affymetrix. After 16 hours of hybridization, hybridization
cocktail-are removed and the DNA array is washed using the a
GeneChip.RTM. Fludics Station (manufactured by Affymetrix) or the
like, and stained with a Streptavidin Phycoerythrin (300 .mu.l of
2.times.MES Stain Buffer (manufactured by Affymetrix), 24 .mu.l of
50 mg/ml acetylated BSA (manufactured by Affymetrix), 6 .mu.l of 1
mg/ml StreptAvidin-Phycoerythr- in (manufactured by Affymetrix),
270 .mu.l of Water (manufactured by Affymetrix)) according to a
Technical Mannual of Affymetrix. There is no particular limitation
for the methods used for hybridization and the method may be
conducted according to the known means.
[0208] (m)-5 Data Analysis
[0209] Data analysis of the DNA array can be carried out using a
commercially available software (f or example, a GCOS (GeneChip
Operating Software) manufactured by Affymetrix; GeneSpring
manufactured by Silicon Genetics; ImaGene manufactured by Takara
Shuzo; Array Gauge manufactured by Fuji Photo Film; ImageQuant
manufactured by Amersham Pharmacia Biotech, or the like) according
to a Technical Manual. Genes which show characteristic expression
profile can be identified and selected for functional analysis.
[0210] Furthermore, the identified gene can be used as a gene
marker to figure out condition of the yeast cells during
fermentation.
[0211] There is no particular limitation for the methods used for
analysis of data and the method may be conducted according to the
known means.
[0212] (n) Classification of Industrial Yeast
[0213] It is possible to classify industrial yeast using a DNA
array mentioned above. Preparation of yeast genomic DNA and
hybridization to a DNA array may be carried out as described
before. Detection of the signal intensity of array is carried out
using a Gene Chip Analysis Basic System and analysis soft ware
(GCOS; GeneChip Operating Software 1.0) manufactured by Affymetrix.
The percentage of probes, to which the DNA of brewing yeast
hybridizes, is calculated and the identity between strain 34/70 and
the tested strain is estimated. Industrial yeast strains can be
classified on the basis of the identity.
[0214] (o) Detection of Nucleotide Polymorphism
[0215] It is possible to detect nucleotide polymorphism of a
industrial yeast by comparative genomic hybridization with the DNA
array mentioned above. The sets of oligonucleotides for each probe
consist of Perfect Match oligonucleotide (PM) which is identical to
the sequence of strain 34/70 and MisMatch oligonucleotide (MM)
which contains a single base mismatch, for example, in the central
position of the oligonucleotide. It is possible to detect
nucleotide polymorphism from the gene whose signal intensity in MM
is higher (for example, more than 5-fold) than that in PM.
[0216] (p) Selection of Genes for Functional Analysis
[0217] From the results of comparative genomic hybridization
analysis, a gene which has probe sets showing low signal
intensities may be lost or have different sequence from that of
strain 34/70. In contrast, a gene which has probe sets showing high
signal intensities may be high in copy number. Such genes can be
selected for functional analysis because the locus may contribute
to the difference of fermentation character between strain 34/70
and the tested strain. The genes which have nucleotide polymorphism
detected by the method mentioned above can be also selected for
functional analysis.
EXAMPLES
[0218] Details of the present invention are mentioned with the
following Examples although the present invention is not limited to
the following Examples.
Example 1
Preparation of Chromosomal DNA of Saccharomyces pastorianus
Weihenstephan 34/70 (Hereinafter, Abbreviated as Strain 34/70)
[0219] Preparation of chromosomal DNA was carried out by a method
mentioned in "Yeast, a practical approach (IRL Press) 6.2.1 (pages
228-229)", which was partially modified. Cells were inoculated and
grown in 200 mL of YPD medium (2% glucose, 1% yeast extract and 2%
polypeptone) at 30.degree. C. until absorbance of the culture at
660 nm became 4. Cells were collected by centrifugation and washed
with buffer A (50 mM sodium phosphate, 25 mM EDTA and 1% (v/v)
.beta.-mercaptoethanol; pH 7.5), resuspended in 25 mL of buffer A,
and 7 mg of Zymolyase 100 T (Seikagaku Kogyo) was added thereto and
the mixture was mildly shaken at 37.degree. C. for 60 minutes. To
this was added 25 mL of buffer B (0.2M Tris-HCl, 80 mM EDTA and 1%
SDS; pH 9.5), then the mixture was allowed to stand at 65.degree.
C. for 30 minutes, cooled on ice, mixed with 12 mL of 5M potassium
acetate and allowed to stand on ice for further 60 minutes. The
resulting solution was centrifuged at 5,000 g for 10 minutes at
15.degree. C. To the recovered supernatant was added the same
volume of ethanol to precipitate DNA, and the mixture was
immediately centrifuged at 5,000 g for 10 minutes at 15.degree. C.
to collect the precipitate. The resulting precipitate was washed
with 70% (v/v) ethanol, subjected to natural drying and dissolved
in 5 mL of TE buffer (10 mM Tris-HCl and 1 mM of EDTA; pH 8.0) to
give a crude DNA solution. Cesium chloride (4.06 g) and 840 .mu.g
of bisbenzimide (Hoechst 33258) were added and dissolved in 3.5 mL
of the crude DNA solution, the mixture was subjected to centrifugal
separation at 100,000 g for 17 hours at 25.degree. C. and exposed
to UV light to make DNA bands visible, whereupon the band of the
lower layer was recovered. The recovered DNA solution was extracted
with isopropanol which was saturated with a cesium chloride
solution to remove bisbenzimide (Hoechst 33258). To the recovered
aqueous layer was added 4-fold by volume of 0.3 M sodium acetate
followed by mixing, and then 3-fold by volume of ethanol was added
thereto to precipitate the DNA, which was recovered by
centrifugation. The recovered DNA was dissolved in TE buffer
containing 75 .mu.g/mL of RNase, kept at 37.degree. C. for 5
minutes, and extracted with phenol/chloroform for three times and
the aqueous layer was further subjected to precipitation with
ethanol. The precipitate recovered by centrifugation was washed
with 70% (v/v) ethanol, subjected to natural drying and dissolved
in TE buffer to prepare a chromosomal DNA solution.
Example 2
Preparation of a Shotgun Library
[0220] The concentration of the genome solution of strain 34/70
prepared in Example 1 was adjusted to 1 mg/mL using a TE buffer and
0.1 mL thereof was treated with a Hydroshear (manufactured by
GeneMachines; speed: 6; cycle: 20) to fragment the genomic DNA. The
ends of the genomic fragment were blunted using a DNA Blunting Kit
(manufactured by Takara Shuzo), fractionated by 0.8% agarose
electrophoresis, and a genomic fragment of 1.5 to 2.5 kb was
excised from the gel and DNA was eluted. The DNA eluate was treated
with phenol/chloroform and precipitated with ethanol to give a
genome library insert. All the above insert and 0.5 .mu.g of pUC 18
SmaI/BAP (manufactured by Amersham Biosciences) were subjected to
ligation at 15.degree. C. for 15 hours using T4 ligase
(manufactured by Takara Shuzo).
[0221] The ligation reaction product was precipitated with ethanol
and dissolved in 10 .mu.L of a TE buffer. A ligation solution (1
.mu.L) was inserted into 40 .mu.L of Escherichia coli Electro Cell
DH5.alpha. (manufactured by Takara Shuzo) by means of
electroporation under the condition mentioned in the attached
experimental manual. The resulting product was spread on an LB
plate medium containing 1.6% of agar (the LB medium (1%
bactotryptone, 0.5% yeast extract and 1% sodium chloride; pH 7.0))
containing 0.1 mg/mL of ampicillin, 0.1 mg/mL of X-gal and 1 mmol/L
of isopropyl-.beta.-D-thiogalactopyranoside (IPTG), and incubated
through the night at 37.degree. C.
[0222] The transformants obtained from colonies formed on the said
plate medium were subjected to cultivation without shaking through
the night at 37.degree. C. in a 384-well titer plate to which 50
.mu.L of an LB medium containing 0.1 mg/mL of ampicillin was added,
and then 50 .mu.L of a 50% aqueous solution of glycerol was added
thereto followed by stirring and the mixture was used as a glycerol
stock.
Example 3
Preparation of a Cosmid Library
[0223] About 0.1 mg of the genome DNA obtained in Example 1 was
partially digested with Sau3AI (manufactured by Takara Shuzo).
Insertion of the fragment into a BamHI site of Super Cos I vector
(manufactured by Stratagene) was carried out according to a manual.
A ligated product prepared by this method was subjected to
packaging using Gigapack III Gold (manufactured by Stratagene) and
introduced into Escherichia coli XL1-Blue MR strain (manufactured
by Stratagene) according to a manual. It was spread on an LB plate
medium containing 0.1 mg/mL of ampicillin and incubated through the
night at 37.degree. C. The resulting transformants were cultured
through the night at 37.degree. C. in an LB medium (each well: 50
.mu.L) containing 0.1 mg/mL of ampicillin using a 96-well titer
plate, and then 50 .mu.L of 50% glycerol solution was added thereto
followed by stirring and the mixture was used as a glycerol
stock.
Example 4
Determination of DNA Sequence
[0224] (4-1) Preparation of DNA Fragment
[0225] The whole genome sequence of strain 34/70 was determined
mainly using the whole genome shotgun method. A DNA fragment of
which DNA sequence is to be determined by that method was prepared
by a PCR method from the shotgun library prepared in the above
Example 2. To be specific, clones derived from the whole genome
shotgun library were inoculated using a replicator (manufactured by
Gene Solution) to a 384-well titer plate where 50 .mu.L of an LB
medium containing 0.1 mg/mL of ampicillin was placed to each well
and cultivated without shaking through the night at 37.degree. C.
The said culture liquid was transferred to a 384-well reaction
plate (manufactured by AB Gene) containing 10 .mu.L of reaction
mixture for PCR (TaKaRa Ex Taq manufactured by Takara Shuzo) using
a replicator (manufactured by Gene Solution) and PCR was carried
out according to a protocol by Makino, et al. "DNA Research, volume
5, pages 1 to 9 (1998)" using a GeneAmp PCR System 9700
(manufactured by Applied Biosystems) to amplify the inserted
fragment. After that, excessive primer and nucleotide were removed
by a PCR product purification kit (manufactured by Amersham
Bioscience) and a sequence reaction was carried out using the
purified PCR sample as a template.
[0226] A DNA fragment from the cosmid library of the above Example
3 was prepared according to the following method. That is, clones
derived from the whole cosmid library were inoculated to each well
of a 96-well plate to which 1.0 mL each of a 2.times.YT medium
(1.6% bactotrypsin, 0.1% yeast extract and 0.5% sodium chloride; pH
7.0) containing 50 g/mL of ampicillin was placed and subjected to
shake culture at 30.degree. C. through the night. A cosmid DNA was
prepared from the said culture using KURABO PI-1100 AUTOMATIC DNA
ISOLATION SYSTEM (manufactured by KURABO) according to a manual of
KURABO, and was used as a template for a sequence reaction.
[0227] (4-2) Sequence Reaction
[0228] A sequence reaction mixture was prepared as follows. The PCR
product or cosmid DNA prepared in the above (4-1) was mixed with
about 21 .mu.l of DYEnamic ET Terminator Sequencing Kit
(manufactured by Amersham Bioscience) and appropriate primers to
give about 8 .mu.l of reaction mixture. An M13 forward (M13-21)
primer and an M13 reverse (M13RV) primer (manufactured by Takara
Bio), were used for the sequence reaction of a PCR product derived
from shotgun DNA, while a forward primer SS-cos F.1 (SEQ ID NO: 7)
and a reverse primer SS-cos R.1 (SEQ ID NO: 8) were used for cosmid
DNA. Amounts of the primer and the DNA fragment were 3.2 pmol and
50 to 200 ng, respectively. The said reaction solution was
subjected to dye terminator sequence reaction of 60 cycles using a
GeneAmp PCR System 9700. Cycle parameter followed a manual attached
to the DYEnamic ET Terminator Sequencing Kit. Purification of the
sample was carried out using a Multi Screen HV Plate (manufactured
by Millipore) according to a manual of Millipore. The purified
reactant was stored in a dark place at 4.degree. C. The said
purified reactant was analyzed using a Mega BACE 1000 Sequencing
System (manufactured by Amersham Bioscience) and ABI PRISM 3700 DNA
Analyser (manufactured by Applied Biosystems) according to manuals
attached thereto. The data on 332,592 sequences obtained by the
Mega BACE 1000 Sequencing System and on 13,461 sequences obtained
by the 3700 DNA Analyser were transferred to a server Enterprise
6500 (manufactured by Sun Microsystems) and preserved. The data on
346,053 sequences corresponded to about 7-fold of the whole genome
size.
[0229] A list of the primers for the PCR used in the Example is
shown in Table 3.
Example 5
Assembly (A Process Whereby the Order of Multiple Sequenced DNA
Fragments is Determined)
[0230] All works for reconstruction of genomic DNA sequence from
sequence information for DNA fragment-of the 346,053 sequences
obtained in the above Example 4 were carried out on a UNIX.RTM.
platform. Base call was carried out by phred (The University of
Washington), masking of vector sequence was carried out using
Cross_Match (The University of Washington) and assembly was carried
out using Phrap (The University of Washington). The contigs
obtained as a result of the assembly were analyzed using a
graphical editor consed (The University of Washington). A series of
works from base call to assembly was carried out all together
utilizing a script phredPhrap attached to the consed.
Example 6
Preparation of a Comparative Database with the Whole Genome
Sequence of S. cerevisiae
[0231] S. pastorianus is believed to be a natural hybrid of S.
cerevisiae with its closely-related species "Int. J. Syst.
Bacteriol., volume 35, pages 508 to 511 (1985)". Therefore, a DNA
sequence (comprising 10,044 bases) of both ends of the cosmid DNA
clone obtained in (4-2) was subjected to a homology searching by a
homology searching algorithm to the genome sequence of S.
cerevisiae, whereupon for each DNA sequence alignment of homologous
region on the genome sequence of S. cerevisiae and the identity
thereof were determined to prepare a database. An identity
distribution chart for cosmid DNA sequence with the corresponding
genomic DNA sequence of S. cerevisiae is shown in FIG. 2. The DNA
sequence of cosmids was roughly classified into a DNA sequence
group showing not less than 94% identity to the genomic DNA
sequence of S. cerevisiae and a DNA sequence group showing
approximately 84% identity thereto. The DNA sequence group showing
not less than 94% identity was named DNA sequence of Sc type
derived from S. cerevisiae and the DNA sequence group showing
approximately 84% identity was named DNA sequence of non-Sc type
derived from genome of closely related species. Similarly, a
comparative database (Table 1) was prepared for the DNA sequence of
both ends of shotgun clone obtained in (4-1) with the genomic DNA
sequence of S. cerevisiae. Table 1 shows an example of the
comparative database of DNA sequence of both ends of 3,648-cosmid
clone with the genomic DNA sequence of S. cerevisiae. Table 1 shows
the homologous region and the identity of forward sequence and
reverse sequence of cosmid subjected to the DNA sequence
determination on each genomic DNA sequence of S. cerevisiae.
1 TABLE 1 Forward Chain Matched S. cerevisiae Genomic Base Sequence
Information Sequence Identical Initiation Termination Length Length
Chromosome Position Position Identity Name of Cosmid (bases)
(bases) No. (bases) (bases) (%) SSL052_A06 627 625 XVI 15,940
16,565 98.7 SSL023_D02 346 341 XVI 16,784 17,125 87.3 SSL015_E09
630 625 XVI 39,030 39,655 89.5 SSL029_B08 664 660 XVI 45,916 45,256
99.3 SSL028_G10 656 655 XVI 47,609 46,954 98.3 SSL008_E01 622 620
XVI 46,362 46,982 93.4 SSL030_G05 632 631 XVI 47,013 47,644 99.2
SSL032_H10 646 645 XVI 52,076 51,431 98.1 SSL041_G05 635 634 XVI
52,979 52,345 99.4 SSL031_D08 659 658 XVI 52,297 52,955 99.2
SSL069_F11 417 414 XVI 55,053 55,467 88.5 SSL005_A10 647 645 XVI
65,233 64,588 99.2 SSL014_G07 628 627 XVI 65,229 65,856 99.8
Reverse Chain Matched S. cerevisiae Genomic Base Sequence
Information Sequence Identical Initiation Termination Length Length
Chromosome Position Position Identity Name of Cosmid (bases)
(bases) No. (bases) (bases) (%) SSL052_A06 626 625 XVI 52,979
52,354 98.7 SSL023_D02 633 629 XVI 66,017 65,388 90.5 SSL015_E09
615 614 XVI 81,655 81,041 97.9 SSL029_B08 650 647 XVI 8,504 9,151
98.8 SSL028_G10 646 641 XVI 10,359 11,000 98.0 SSL008_E01 589 587
XVI 86,022 85,435 98.3 SSL030_G05 618 617 XVI 87,004 86,387 99.5
SSL032_H10 637 636 XVI 13,273 13,909 98.7 SSL041_G05 619 618 XVI
9,825 10,443 99.4 SSL031_D08 638 637 XVI 92,295 91,658 99.1
SSL069_F11 788 787 XVI 97,115 96,328 94.4 SSL005_A10 527 516 XVI
21,537 22,053 81.8 SSL014_G07 621 620 XVI 103,674 103,054 99.2
[0232] On the basis of the information obtained by the prepared
comparative database, mapping of cosmid clone and shotgun clone on
S. cerevisiae genome sequence was carried out (FIG. 3). In
addition, a comparative database (Table 1) of contig DNA sequence
obtained in Example 5 with S. cerevisiae genome sequence was
prepared, then mapping was carried out. Although the means for the
mapping was almost the same as the above-mentioned method, if
forward and reverse sequence of cosmid and shotgun clones were
present in different contigs, these contigs wewe connected by
forward-reverse link (FIG. 4).
Example 7
Identification and Assingment of Function of ORF
[0233] Identification of ORF (open reading frame) in the DNA
sequence assembled in Example 5 was carried out. The examples are
specifically shown below. Identification of ORF existing in the DNA
sequence assembled in Example 5 was carried out using a available
program using ORF finder (http://www.ncbi.nih.gov/gorf/gorf.html)
for identification of ORF for six kinds of reading frames in the
sequence with the length of not less than 150 bases from initiation
codon to termination codon including its complementary sequence.
Assignment of function of the extracted ORF was carried out by
homology searching of amino acid sequence of ORFs of S. cerevisiae
that have been registered at the SGD and published. Table 2 shows
examples of the ORF name of S. cerevisiae corresponding to the
result of assignment of function of ORF existing in the non-Sc
genome. From the left side of the table, name of the ORF existing
on the brewing yeast, ORF length in polynucleotide, ORF length in
polypeptide, name of the ORF of S. cerevisiae determined by
homology searching, identity, coincided length and functions of the
gene are shown.
2TABLE 2 ORF ORF Name of Coincided Length Length Homologous
Identity Length Name of ORF (bp) (aa) Gene (%) (aa) Functions
nonSc-ATF2 1638 545 ATF2 71 535 alchhol O-acetyltransferase
nonSc-THI3 1305 434 THI3 94 431 transcriptional activator
nonSc-FUS3 435 144 FUS3 90 139 MAP kinase nonSc-ILV5 1188 395 ILV5
97 395 ketol-acid reductoisomerase nonSc-MET2 1461 486 MET2 93 486
homoserine O-acetyltransferase nonSc-MET10 3108 1035 MET10 87 1035
sulfite reductase (NADPH) nonSc-MET14 609 202 MET14 97 202
adenylsulfate kinase nonSc-MET16 786 261 MET16 92 261
phosphoadenylyl-sulfate reductase nonSc-TPI1 747 248 TPI1 96 248
triosephosphate isomerase nonSc-MET3 1536 511 MET3 94 511 sulfate
adenylyltransferase (ATP) nonSc-MET10 3108 1035 MET10 87 1035
sulfite reductase (NADPH) nonSc-SAM1 1149 382 SAM1 97 382
methionine adenosyltransferase nonSc-SSU1 1377 458 SSU1 78 457
sulfite transporter
Example 8
Analysis of Chromosome Structure by DNA Microarray-Based
Comparative Genomic Hybridization and PCR
[0234] Preparation of genomic DNA from yeast was carried out using
Qiagen Genomic Tip 100/G (#10243: manufactured by Qiagen) and
Qiagen Genomic DNA Buffer Set (#19060: manufactured by Qiagen)
according to the manuals attached to the kits. The DNA (10 .mu.g)
was digested with DNase I (manufactured by Invitrogen) according to
a method of Winzeler, et al. 37 Science, volume 281, pages 1194 to
1197 (1998)", biotinylated by a terminal transferase (manufactured
by Roche) and hybridized to a DNA microarray (Affymetrix Gene Chip
Yeast Genome S98 Array: produced by Affymetrix). Hybridization and
detection of signal intensity of array were carried out using a
Gene Chip Analysis Basic System manufactured by Affymetrix.
[0235] The signal of each probe hybridized with the DNA of strain
34/70 is normalized to that of the haploid laboratory yeast strain
S288C using an analysis soft ware (Microarray Suite 5.0:
manufactured by Affymetrix) and shown as signal log ratio
(2.sup.n). Signal log ratios were lined following genes order in
each chromosome using a spreadsheet program (Microsoft Excel 2000)
and the signal log ratios are shown in bar graphs as shown in FIG.
5. The non-Sc type genes do not hybridize to the S. cerevisiae
array, therefore, the Sc type gene dosage affect the signal log
ratio and the points where the signal log ratios show vigorous
changes are considered to be translocation sites between Sc type
and non-Sc type chromosome.
[0236] On the basis of genome sequence of strain 34/70 determined
by a shotgun method, the chimera chromosome structure was confirmed
by PCR where two pairs of primers having DNA sequences in which one
side is Sc type while the other side is a non-Sc type
(XVI-1(L)cer-95894 (SEQ ID NO: 9)/XVI-1(R)nonSc-106302rv (SEQ ID
NO: 10) and XVI-2(L)cer-859737 (SEQ ID NO:
11)/XVI-2(R)nonSc-864595rv (SEQ ID NO: 12) were designed and the
genomic DNA derived from strain 34/70 was used as a template. Two
examples of translocation of chromosome XVI are shown as
follows.
[0237] The PCR was carried out using Takara LA Taq.TM. and a buffer
attached thereto in accordance with the attached manual by a Takara
PCR Thermal Cycler SP.
[0238] As a result of the PCR, it was confirmed by a 0.8% agarose
electrophoresis that, a DNA fragment in the predicted length was
amplified from strain 34/70, while when genomic DNA of the
experimental strain S. cerevisiae X2180-1A was used as a template
for the PCR, amplification of the DNA fragment was not detected.
Furthermore, when DNA sequence of both ends of the DNA fragment
amplified from strain 34/70 was confirmed, it was consistent with
the genome sequence determined by a shotgun method and it was
confirmed that, within such a region, translocation between Sc type
and non-Sc type chromosome took place.
[0239] From the above result, it is estimated that at least two
kinds of chromosomes were present in the chromosome XVI as shown in
FIG. 6. According to the same technique, ligation between Sc
chromosome and non-Sc chromosome (or inverse thereof) or, in other
words, the region where the existence of chimera chromosome
structure was suggested was confirmed. Such chimera chromosome
structure of the Sc chromosome and non-Sc chromosome was confirmed
in at least 13 places in the total chromosomes of strain 34/70
(FIG. 1).
[0240] As a result of genome analysis, it was found that chromosome
structure of bottom fermenting yeast was very complicated and there
were at least 0.37 kinds of chromosomes in strain 34/70.
Example 9
Cloning of SSU1 Genes of Strain 34/70
[0241] The shotgun clone containing non-ScSSU1 gene was retrieved
using a comparative database obtained in Example 6. There was
SSS103_G08 which contained about 2.4 kb of fragment containing
full-length of non-ScSSU1 ORF, where identity of forward and
reverse sequence of shotgun clone to those of S. cerevisiae were
62.9% and 82.9%, respectively.
[0242] SSS103-G08 was selected from a library of genomic DNA, then
full length of non-ScSSU1 was prepared by PCR. Synthetic DNAs of
SacI-non-Sc-SSU1_for1 (SEQ ID NO: 13) and BglII-non-Sc-SSU1_ry1460
(SEQ ID NO: 14) were used as primers. As a result of such a
combination, base numbers 1 to 1460 of nonScSSU1 was amplified to
give a SacI-BglII fragment of about 1.5 kb.
[0243] With regard to an ScSSU1 gene, the full length gene was
obtained by PCR using a primer pair designed on the basis of the
information of SGD using the genomic DNA of strain 34/70 as a
template. Synthetic DNAs of SacI-ScSSU1_for1 (SEQ ID NO: 15) and
BglII-ScSSU1_ry1406 (SEQ ID NO: 16) were used as primers. As a
result of such a combination, base numbers 1 to 1406 of ScSSU1 gene
was amplified to give a SacI-BglII fragment of about 1.4 kb.
[0244] ScSSU1 and non-ScSSU1 genes obtained as above were inserted
using TA cloning kit (Invitrogen) into pCR 2.1-TOPO vector attached
to the kit, and they were named TOPO/ScSSU1 and TOPO/non-ScSSU1,
respectively. Sequences of the resulting ScSSU1 and non-ScSSU1
genes were confirmed by a method of Sanger "F. Sanger, Science,
volume 214, page 1215, 1981" (FIG. 10).
Example 10
Disruption of Each SSU1 Gene
[0245] According to a method mentioned in the document "Goldstein,
et al., Yeast, volume 15, page 1541 (1999)", DNA fragments for gene
disruption were prepared by PCR using a plasmid containing a
drug-resistance marker (pFA6a (G418.sup.r), pAG 25 (nat1)) as a
template. As a primer for the PCR, non-Sc-SSU1_for (SEQ ID NO:
17)/non-Sc-SSU1_rv (SEQ ID NO: 18) was used for disruption of
non-ScSSU1 gene, while for disruption of ScSSU1 gene, ScSSU1_for
(SEQ ID NO: 19)/ScSSU1_rv (SEQ ID NO: 20) was used. For disruption
of non-ScSSU1 gene, a plasmid pPGAPAUR (AUR1-C) and a primer
non-Sc-SSU1_for +pGAPAUR (SEQ ID NO: 21)/non-Sc-SSU1_rv+AUR1-C (SEQ
ID NO: 22) were further used. As such, two and three kinds of DNA
fragments were prepared for ScSSU1 and non-ScSSU1 gene disruption,
respectively.
[0246] The bottom fermenting yeast BH 96 was transformed using the
DNA fragment for gene disruption prepared with the method above.
The transformation was carried out by a method mentioned in the
Japanese Patent Laid-Open Gazette No. 07/303,475 and concentrations
of the drugs were 300 mg/L for geneticin, 50 mg/L for
nourseothricin and 1 mg/L for aureobasidin A.
[0247] With regard to the transformants prepared, gene disruption
was confirmed by Southern analysis. Firstly, the genomic DNA
extracted from parental strain and disruptant was subjected to
restriction enzyme treatment (at 37.degree. C. or 18 hours) using
NcoI for the confirmation of ScSSU1 gene disruption and HindIII for
the confirmation of non-ScSSU1 gene disruption, and then
fractionated by 1.5% agarose gel electrophoresis and transferred to
a membrane. After that, hybridization was carried out (at
55.degree. C. for 18 hours) with a probe specific to the ScSSU1 or
non-ScSSU1 following a protocol of the Alkphos Direct Labelling
Reagents (Amersham) and signals were detected by CDP-Star.
[0248] Each of the strains where gene disruption was confirmed was
named as follows.
[0249] Sc-1 (ScSSU1/Scssul::G418r)
[0250] Sc-2 (Scssu1::G418.sup.r/Scssu1::nat1)
[0251] non-Sc-1 (non-ScSSU1/non-ScSSU1/non-Scssu1::G418.sup.r)
[0252] non-Sc-2
(non-ScSSU1/non-Scssul::G418.sup.r/non-Scssu1::nat1)
[0253] non-Sc-3
(non-Scssu1::G418.sup.r/non-Scssu1::nat1/non-Scssu1::AUR1-- C)
Example 11
Quantitative Analysis of Sulfite Production in a Fermentation
Test
[0254] Fermentation test using parental strain and disruptant Sc-1
to non-Sc-3 prepared in Example 10 was carried out under the
following condition.
[0255] Original extract: 12.75%
[0256] Fermentation scale: 2 liters
[0257] Dissolved oxygen concentration: about 9 ppm
[0258] Fermentation temperature: 15.degree. C.
[0259] Pitching rate: 10 g of wet yeast cells/2 L of wort
[0260] Wort was periodically sampled and monitored in cell growth
(OD 600) (FIG. 7-(a)), apparent extract (FIG. 7-(b)) and sulfite
concentration (FIG. 7-(c)). Quantitative analysis of sulfite in
wort was carried out in such a method by which sulfite is captured
in a hydrogen peroxide solution by means of distillation in an
acidic condition and subjected to titration with an alkali (Revised
Method for BCOJ Beer Analysis by the Brewing Society of Japan).
[0261] As a result, sulfite production in the wort by ScSSU1
disruptant was nearly the same as that produced by the parental
strain, while it significantly decreased by non-ScSSU1 disruptant.
It was suggested that non-ScSSU1 gene which is specific to bottom
fermenting yeast greatly contributes to sulfite production in
wort.
[0262] At the same time, growth rate and extract-consuming rate
were significantly decreased in the non-ScSSU1 disruptant, and it
supported that excessive sulfite in cells causes inhibition of cell
growth.
Example 12
Overexpression of Each SSU1 Gene
[0263] From the plasmid TOPO/non-ScSSU1 mentioned in Example 9, a
fragment of about 1.5 kb including the full length of non-ScSSU1
ORF was excised by a treatment with restriction enzymes
(SacI-BglII). Then this fragment was inserted into a plasmid
pNI-NUT which was similarly treated with restriction enzymes
(SacI-BglII) to construct a non-ScSSU1 overexpression vector
pYI-non-ScSSU1. The vector pNI-NUT contains URA3 as a homologous
recombination site and nourseothricin-resistance gene (nat1) and
ampicillin-resistance gene (Amp.sup.r) as selective markers. On the
other hand, the ScSSU1 overexpression vector pNI-ScSSU1 has a
structure where the non-ScSSU1 gene of the above-mentioned
pYI-non-ScSSU1 is substituted with the SSU1-R of about 2 kb derived
from S. cerevisiae "J. Ferment. Bioeng., volume 86, page 427
(1998)". For overexpression of each SSU1 gene, promoter and
terminator of glyceraldehyde-3-phosphate dehydrogenase gene (TDH3)
were used.
[0264] Bottom fermenting yeast BH225 was transformed by a
overexpression vector prepared following the above-mentioned
method. Transformation was carried out by a method mentioned in the
Japanese Patent Laid-Open Gazette No. 07/303,475 and selected on
YPD plate medium containing 50 mg/L of nourseothricin.
[0265] Confirmation of the overexpression was carried out by
RT-PCR. Extraction of total RNA was carried out using an RNeasy
Mini Kit (Qiagen), according to the manual of "for total RNA
isolation from yeast" attached the kit. ScSSU1_for331 (SEQ ID NO:
23)/ScSSU1.sub.--982rv (SEQ ID NO: 24) were used as ScSSU1-specific
primers; non-ScSSU1_for329 (SEQ ID NO: 25)/non-ScSSU1.sub.--981rv
(SEQ ID NO: 26) were used as non-ScSSU1-specific primers; and
PDA1_for1 (SEQ ID NO: 27)/PDA1.sub.--730rv (SEQ ID NO: 28) were
used as specific primers for constitutively expressed gene PDA1
used as an internal standard. PCR product was fractionated by 1.2%
agarose electrophoresis, stained with an ethidium bromide solution
and signal value of each SSU1 gene of transformant was normalized
with a signal value of PDA 1 and compared with that of the parental
strain. The overexpressed strains confirmed as such, were named as
ScSSU1 overexpressed strain and non-ScSSU1 overexpressed
strain.
Example 13
Quantitative Analysis of Sulfite Production in a Fermentation
Test
[0266] Fermentation tests using parental strain and each of the
SSU1 overexpressed strains obtained in the Example 12 were carried
out under the following condition.
[0267] Original extract: 12.83%
[0268] Fermentation scale: 2 liters
[0269] Dissolved oxygen concentration: about 9 ppm
[0270] Fermentation temperature: 12.degree. C.
[0271] Pitching rate: 10 g of wet yeast cells/2 L of wort
[0272] As in Example 11, Wort was periodically sampled and
monitored in cell growth (OD 600) (FIG. 8-(a)), apparent extract
(FIG. 8-(b)) and sulfite concentration (FIG. 8-(c)). With regard to
the sulfite production, it was only slightly higher in Sc SSU1
overexpressed strain (19 ppm at the end of the fermentation) as
compared with that of the parental strain (12 ppm at the same
stage), while non-Sc SSU1 over expressed strain showed a
significant increase (45 ppm at the same stage). At the same time,
there was no difference in the growth rate and in the
extract-consuming rate between the parental strain and the
overexpressed strains.
[0273] From the above result, by overexpression of the gene
encoding the sulfite-discharging pump specific to the bottom
fermenting yeast shown in the present invention, it is possible to
increase sufite concentration in beer without changing the
fermentation process and the fermentation period. As a result, it
is now possible to produce an alcoholic beverage with excellent
flavor stability and a longer quality preservation period.
Example 14
Cloning of MET14 Gene of Strain 34/70
[0274] DNA sequence of non-Sc MET14 gene was retrieved from the
comparative database obtained in Example 6. A shotgun clone SSS
134.sub.--021 containing about 1.9 kb (full-length) of non-Sc MET14
gene was obtained; its forward and reverse DNA sequence identity to
S. cerevisiae were 79.0% and 56.0%, respectively.
[0275] The shotgun clone 134.sub.--021 was selected from a shotgun
library and the full length non-Sc MET14 gene was obtained by PCR.
As a primer pair, synthetic DNAs of SacI-nonSc-MET14_for-21 (SEQ ID
NO: 29) and BamHI-nonSc-MET14_rv618 (SEQ ID NO: 30) were used
(Table 3). As a result of such a combination, a non-Sc MET14 gene
(about 0.6 kb) embraced by SacI and BamHI restriction sites was
obtained.
3TABLE 3 SEQ ID No Sequence Name 5'-Base Sequence-3' 5 M13_for
agtcacgacg ttgta 6 M13_rv caggaaacag ctatgac 7 SS-cosF.1 aggcgtatca
cgaggccctt tc 8 SS-cosR.1 cttatcgatg ataagcggtc aaacatga 9 XVI-1
(L) cer-95894 cgcaagctcc gtacgttcaa cattcttatg aacggc 10 XVI-1 (R)
nonSc-106302rv gcatcatcgt cgtgatcctt ctttggcaaa tgcagg 11 XVI-2 (L)
cer-859737 gcgggtattt tgatggtaaa tctacaagcc ctcggc 12 XVI-2 (R)
nonSc-864595rv cccagacaca gtttccagta tcatcctcgc agaac 13
SacI-nonScSSU1_for1 gagctcatgg tcgctagttg gatgct 14
BglII-nonScSSU1_rv1460 agatctcagc ttcagcccaa tccatt 15
SacI-ScSSU1_for1 gagctcatgg ttgccaattg ggtact 16
BglII-ScSSU1_rv1406 agatctctcc tacatgaaat gcttgc 17 nonScSSU1_for
atggtcgcta gttggatgct cactgccaca agggatttca accctttcat atcgaatatt
ctgtacagct gtttgtcatg gttatggggg tcggtatttc ccttgacagt cttgacgtgc
18 nonScSSU1_rv tgttaaatat gtactatcga tagccgagtt tgattcctcc
acactttcga acagtcttct ccgtcccttc ctctgataaa tgctgttgaa aggagaattg
cgcacttaac ttcgcatctg 19 ScSSU1_for atggttgcca attgggtact
tgctcttacg aggcagtttg accccttcat gtttatgatg gtcatgggtg tcggcatttc
atcgaatatt ctatatagct ccttgacagt cttgacgtgc 20 SuSSU1_rv ttatgctaaa
cgcgtaaaat ctagagccga gtttgattct tccacgcttt caatgctgtt atacggagaa
actgtcgtct tttccgtacc tgactctgaa cgcacttaac ttcgcatctg 21
nonScSSU1_for + pGAPAUR atggtcgcta gttggatgct cactgccaca agggatttca
accctttcat gtttgtcatg gttatggggg tcggtatttc atcgaatatt ctgtacagct
ccggagctta ccagttctca 22 nonScSSU1_rv + AUR1-C tgttaaatat
gtactatcga tagccgagtt tgattcctcc acactttcga tgctgttgaa aggagaattg
acagtcttct ccgtcccttc ctctgataaa tcgactctag aggatucaga 23
ScSSU1_for331 tcgaaagcga acacgacgaa 24 ScSSU1_982rv cgacagaaat
cacggtgaaa a 25 nonScSSU1_329 tgtcacaaaa atttaccacg ac 26
nonScSSU1_981rv aagggaaatt accgtaaaga ag 27 PDA1_for1 atgtttgtcg
cacctgtatc t 28 PDA1_730rv gattagaggc accatcac 29
SacI-nonSc-MET14_for-21 ctcgagctct cgtgaaattc attgaaacaa atg 30
BamHI-nonSc-MET14_rv618 ggatccttat aagatttata gatgcttccg 31
SacI-ScMET14_for ctcgagctca gaaaagttgg aattatttct cca 32
BamHI-ScMET14_rv ggatccaatg tacagtaatc ggtcaaatta
[0276] With regard to an Sc MET14 gene, a full length of the
structural gene was obtained by PCR using a primer pair designed on
the basis of the information of SGD and using genomic DNA of strain
34/70 as a template. Synthetic DNAs of SacI-ScMET14_for (SEQ ID NO:
31) and BamHI-ScMET14_rv (SEQ ID NO: 32) were used as primers. As a
result of such a combination, a Sc MET14 gene (about 0.6 kb)
embraced by SacI and BamHI restriction sites was obtained.
[0277] The Sc MET14 and non-Sc MET14 genes obtained as above were
inserted using a TA cloning kit (manufactured by Invitrogen) into
pCR 2.1-TOPO vector attached to the kit, and they were named
TOPO/ScMET14 and TOPO/nonSc-MET14, respectively.
[0278] DNA sequences of the resulting Sc MET14 and non-Sc MET14
genes were checked by a method by Sanger "Science, volume 214, page
1215 (1981)" (FIG. 11).
Example 15
Overexpression of Each MET14 Gene in Sc SSU1 Overexpressed
Strain
[0279] A fragment of about 0.6 kb containing Sc MET14 or non-Sc
MET14 mentioned in Example 14 was inserted into the multi-cloning
site of the expression vector pUP3GLP (Japanese Patent Laid-Open
Gazette No. 2000/316,559) to construct overexpression vectors
pUP3Sc MET14 and pUP3nonSc-MET14 in which each MET14 gene was
expressed under control of glyceraldehyde-3-phosphate dehydrogenase
promoter and terminator. Top fermenting yeast, strain KN009F, was
transformed by an Sc SSU1 overexpression vector pNI-SSU1 mentioned
in Example 12 to prepare strain FOY227 which is an Sc SSU1
overexpressed strain. Strain FOY227 was transformed by the above
pUP3ScMET14 and pUP3nonSc-MET14 to prepare strain FOY306 and strain
FOY 307 in which Sc MET14 and non-Sc MET14, together with Sc SSU1,
are overexpressed, respectively.
Example 16
Quantitative Analysis of Sulfite Production in a Fermentation
Test
[0280] Fermentation tests were carried out using strains prepared
in Example 15; strain FOY227 which is an Sc SSU1 overexpressed
strain, strain FOY306 which is an Sc MET14 overexpressed strain in
strain FOY227, strain FOY307 which is a non-Sc MET14 overexpressed
strain in strain FOY227 and the parental strain KN009 F under the
following condition.
[0281] Original extract: 12.84%
[0282] Fermentation scale: 1.5 liters
[0283] Dissolved oxygen concentration: about 9 ppm
[0284] Fermentation temperature: 25.degree. C. at all times
[0285] Pitching rate: 7.5 g of wet yeast cells/1.5 L of wort
[0286] As in Example 11, wort was periodically sampled and
monitored in cell growth (OD 600), apparent extract and sulfite
concentration. With regard to the yeast growth and the consumed
amount of extract, there was no difference among the strains.
However, with regard to the sulfite production, it was only
slightly higher in Sc SSU1 overexpressed strain FOY227 (3.4 ppm at
the end of the fermentation), and Sc MET14 and Sc SSU1
overexpressed strain FOY306 (6.4 ppm at the same stage) as compared
with that of the parental strain KN009F (0.32 ppm at the same
stage), while non-Sc MET14 and Sc SSU1 overexpressed strain FOY307
showed a significant increase (16.6 ppm at the same stage) as shown
in FIG. 9.
[0287] From the above results, it was found that overexpression of
the gene encoding the adenylyl sulfate kinase specific to the
bottom fermenting yeast shown in the present invention was
effective to increase sufite concentration in beer without changing
the fermentation process and the fermentation time. As a result, it
is now possible to produce an alcoholic beverage with excellent
flavor stability and a longer quality preservation period.
Example 17
Production of the Bottom Fermenting Yeast DNA Microarray
[0288] DNA microarray of bottom fermenting yeast was produced based
on the DNA sequence information of the ORFs obtained in the above
(h) and intergenic DNA sequences located between ORFs deduced from
whole genome sequence of strain 34/70.
[0289] Production of the DNA Microarray
[0290] Based on the DNA sequence information of the following four
groups; (1) 22483 regions from the whole genome sequence
information of 34/70 strain, (2) 403 S. cerevisiae ORFs from SGD
which are not identified as Sc type ORFs in 34/70 strain, (3) 27
regions from S. pastorianus genes submitted in Genbank, (4) 64 DNA
sequences of genes used as internal standard, PM probes (Perfect
Match Probe; 25 base long) which are unique against whole genome
sequence of the bottom fermenting yeast were designed using
GeneChip.RTM. (Affymetrix) technology.
[0291] In order to obtain quantitative and reproducible
information, 11 probes and 20 probes were designed for each locus
or region of (1), (2), (3) and (4) respectively. To further
increase the sensitivity and specificity of the detection, mismatch
probes (MM probe) that have sequences identical to the PM probe
with the exception of one mismatched base at the central position
(i.e. base 13) was also designed.
[0292] All of designed PM and MM probes were synthesized and packed
in the glass slide (manufactured by Affymetrix) to produce the
microarray using GeneChip.RTM. technology.
[0293] (1) was comprised in;
[0294] A) 6307 DNA sequences of non-Sc type ORFs, B) 7640 DNA
sequences of Sc type ORFs, C) 28 DNA sequences of mitochondrial
ORFs from 34/70 strain, D) 553 DNA sequences which have not been
identified as the above ORFs but have some similarity to the
proteins of S. cerevisiae using NCBI-BlastX homology searching, E)
7955 intergenic DNA sequences between as above A) or B).
[0295] (2) was comprised in;
[0296] YBL108C-A, YBR074W, YFL061W, YIL165C, YGR291C, YJR052W,
YDR223W, YAL025C, YAR073W, YFL057C, YLL015W, YJR105W, YLR299C-A,
YNR073C, YDL246C, YHL049C, YAR010C, YKL096W, YBL026W, YMR230W,
YAL037C-A, YAL037C-B, YAL037W, YAL063C-A, YAL064C-A, YAL064W,
YAL065C, YAL068W-A, YAL069W, YAR009C, YAR020C, YAR042W, YAR047C,
YAR053W, YAR060C, YAR061W, YAR062W, YBL027W, YBL040C, YBL068W-A,
YBL101W-B, YBL109W, YBL112C, YBR092C, YBR191W-A, YBR219C, YCL019W,
YCL029C, YCL065W, YCL066W, YCL068C, YCL069W, YCL073C, YCL074W,
YCL075W, YCL076W, YCR035C, YCR036W, YCR038W-A, YCR10 1C, YCR104W,
YCR105W, YCR106W, YCR107W, YCR108C, YDL003W, YDL037C, YDL064W,
YDL073W, YDL094C, YDL095W, YDL096C, YDL136W, YDL143W, YDL152W,
YDL191W, YDL200C, YDL201W, YDL247W-A, YDL248W, YDR014W, YDR015C,
YDR034C-D, YDR039C, YDR045C, YDR098C-B, YDR160W, YDR210C-D,
YDR210W-B, YDR215C, YDR225W, YDR261C-D, YDR261W-B, YDR292C,
YDR302W, YDR304C, YDR305C, YDR342C, YDR344C, YDR364C, YDR365W-B,
YDR427W, YDR433W, YDR471W, YDR510C-A, YDR543C, YDR544C, YEL012W,
YEL075W-A, YER039C-A, YER046W-A, YER056C-A, YER060W-A, YER074W,
YER138C, YER187W, YER188C-A, YER190C-A, YFL002W-A, YFL014W,
YFL019C, YFL020C, YFL030W, YFL031W, YFL051C, YFL052W, YFL053W,
YFL054C, YFL055W, YFL056C, YFL063W, YFL065C, YFL066C, YFL067W,
YFR012W-A, YGL028C, YGL041C, YGL052W, YGL210W-A, YGL259W, YGL262W,
YGL263W, YGR034W, YGR038C-B, YGR089W, YGR107W, YGR109W-A,
YIL082W-A, YGR122C-A, YGR146C, YGR148C, YGR161W-B, YGR182C,
YGR183C, YGR271C-A, YGR290W, YGR295C, YHL009W-A, YHL009W-B,
YHL015W-A, YHL046W-A, YHL047C, YHL048C-A, YHL048W, YHR032C-A,
YHR032W-A, YHR039C-A, YHR043C, YHR070C-A, YHR071C-A, YHR071W,
YHR141C, YHR165W-A, YHR179W, YHR180C-B, YHR180W-A, YHR182W,
YHR193C, YHR193C-A, YHR207C, YHR211W, YHR213W-A, YHR216W, YHR217C,
YHR218W-A, YIL029C, YIL052C, YIL069C, YIL148W, YIL171W, YIL174W,
YIL176C, YIR018C-A, YIR041W, YIR042C, YIR043C, YIR044C, YJL012C-A,
YJL014W, YJL062W-A, YJL136C, YJL175W, YJL222W-B, YJR024C, YJR027W,
YJR032W, YJR053W, YJR054W, YJR094W-A, YJR107W, YJR110W, YJR111C,
YJR140W-A, YJR151C, YJR152W, YJR153W, YJR154W, YJR155W, YJR162C,
YKL018W, YKL020C, YKL044W, YKL224C, YKL225W, YKR012C, YKR013W,
YKR017C, YKR018C, YKR019C, YKR020W, YKR035C, YKR036C, YKR040C,
YKR041W, YKR042W, YKR052C, YKR053C, YKR057W, YKR062W, YKR094C,
YKR102W, YKR103W, YKR104W, YLL014W, YLL030C, YLL037W, YLL038C,
YLL043W, YLL065W, YLR029C, YLR030W, YLR062C, YLR098C, YLR099W-A,
YLR107W, YLR139C, YLR140W, YLR142W, YLR144C, YLR145W, YLR154C-G,
YLR154W-A, YLR154W-B, YLR154W-C, YLR154W-E, YLR154W-F, YLR155C,
YLR156W, YLR157C-B, YLR157W-C, YLR162W, YLR205C, YLR207W, YLR209C,
YLR227W-B, YLR236C, YLR237W, YLR238W, YLR245C, YLR251W, YLR271W,
YLR278C, YLR287C-A, YLR305C, YLR306W, YLR311C, YLR317W, YLR338W,
YLR344W, YLR345W, YLR354C, YLR364W, YLR380W, YLR401C, YLR402W,
YLR410W-B, YLR411W, YLR412C-A, YLR412W, YLR413W, YLR448W, YLR460C,
YLR461W, YLR463C, YLR465C, YML003W, YML039W, YML073C, YMR087W,
YMR143W, YMR175W-A, YMR247W-A, YMR268W-A, YMR324C, YMR325W,
YNL020C, YNL035C, YNL054W-B, YNL243W, YNR034W-A, YNR075C-A,
YNR077C, YOL038C-A, YOL053W, YOL101C, YOL103W-B, YOL162W, YOL163W,
YOL164W, YOL164W-A, YOL165C, YOL166C, YOL166W-A, YOR050C, YOR096W,
YOR101W, YOR192C-B, YOR192C-C, YOR225W, YOR235W, YOR343W-B,
YOR366W, YOR381W-A, YOR382W, YOR383C, YOR384W, YOR385W, YOR386W,
YOR387C, YOR389W, YPL003W, YPL019C, YPL023C, YPL036W, YPL048W,
YPL055C, YPL060C-A, YPL175W, YPL194W, YPL197C, YPL257W-B,
YPR002C-A, YPR008W, YPR014C, YPR028W, YPR043W, YPR048W, YPR087W,
YPR094W, YPR108W, YPR137C-B, YPR161C, YPR162C, YPR163C, YPR164W,
YPR165W, YPR166C, YPR167C, YPR168W, YPR169W, YPR169W-A, YPR170C,
YPR170W-A, YPR171W, YPR172W, YPR173C, YPR174C, YPR175W, YPR176C,
YPR177C, YPR178W, YPR179C, YPR180W, YPR181C, YPR182W, YPR183W,
YPR184W, YPR185W, YPR186C, YPR187W, YPR188C, YPR189W, and
YPR190C
[0297] (3) was comprised in;
[0298] GenBank Accession No. AY130327, BAA96796.1, BAA96795.1,
BAA14032.1, NP.sub.--012081.1, NP.sub.--009338.1, BAA19915.1,
P39711, AY130305, AF399764, AX684850, AB044575, AF114923, AF114915,
AF114903, M81158, AJ229060, X12576, X00731, X01963
[0299] (4) was comprised in:
[0300] GenBank Accession No. J04423.1, J04423.1, J04423.1,
J04423.1, J04423.1, J04423.1, J04423.1, X03453.1, X03453.1,
L38424.1, L38424.1, L38424.1, X17013.1, X17013.1, X17013.1,
M24537.1, M24537.1, M24537.1, X04603.1, X04603.1, X04603.1,
K01391.1, K01391.1, K01391.1, J04423.1, J04423.1, J04423.1,
J04423.1, J04423.1, J04423.1, J04423.1, X03453.1, X03453.1,
L38424.1, L38424.1, L38424.1, X17013.1, X17013.1, X17013.1,
M24537.1, M24537.1, M24537.1, X04603.1, X04603.1, X04603.1,
V01288.1, V01288.1, V01288.1, X16860.1, X16860.1, X16860.1,
L12026.1, L12026.1, L12026.1, Z75578.1, Z75578.1, Z75578.1,
Z75578.1, Z75578.1, J01355.1, J01355.1, J01355.1, J01355.1 and
J01355.1
Example 18
Identification of Molecular Markers that were Highly Inducible in
Zinc Depleted Condition.
[0301] 1. Preparation of mRNA
[0302] Strain 34/70 was grown overnight in LZMM medium+40 .mu.M
zinc sulfate at 30.degree. C. with shaking. LZMM medium contains
0.17% yeast nitrogen base w/o amino acids (manufactured by DIFCO),
0.5% ammonium sulfate, 20 mM sodium citrate (pH 4.2), 125 .mu.M
MnCl2, 10 .mu.M FeCl2, 2% maltose, 10 mM EDTA (pH 8.0). Cells were
harvested and washed three times with sterile distilled water
before inoculation to an optical density (OD600) of 0.25 in 500 ml
of 1) zinc depleted medium (LZMM medium), 2) zinc replete medium
(LZMM+40 .mu.M zinc sulfate), 3) oxidative stress medium (LZMM+40
.mu.M zinc sulfate+2 mM H.sub.2O.sub.2), 4) carbon starvation
medium (deleting maltose from above LZMM+40 .mu.M zinc sulfate).
Cells were grown at 30.degree. C. for 6 hours and harvested for RNA
preparation.
[0303] Preparation of total RNA was carried out using RNeasy.RTM.
Mini Kit (manufactured by QIAGEN) according to the attached manual.
Preparation of Poly(A)+ mRNA from total RNA was carried out using
Oligotex Direct mRNA kit (manufactured by QIAGEN) according to the
attached manual.
[0304] 2. Synthesis of Biotin-Labeled cRNA
[0305] Synthesis of Biotin-Labeled CRNA was carried out using
BioArray HighYield RNA Transcript Labeling Kit (manufactured by
Affymetrix) according to the attached manual.
[0306] 3. Hybridization
[0307] 5 .mu.g of Biotin-Labeled cRNA, 1.7 .mu.l of 3 nM Control
Oligonucleotide B2 (manufactured by Affymetrix), 5 .mu.l of
20.times. Eukaryotic Hybridization Controls (manufactured by
Affymetrix), 1 .mu.l of 10 mg/ml Herring Sperm DNA (manufactured by
Affymetrix), 1 .mu.l of 50 mg/ml Acetylated BSA (manufactured by
Affymetrix), 50 .mu.l of 2.times. Hybridization buffer
(manufactured by Affymetrix), and water (manufactured by
Affymetrix) to give final volume of 100 .mu.l were mixed and
hybridized to the DNA microarray according to a Technical Mannual
of Affymetrix. After 16 hours of hybridization, hybridization
cocktail was removed and the DNA microarray was washed using the
GeneChip.RTM. Fludics Station (manufactured by Affymetrix), and
stained with 600 .mu.l of Streptavidin Phycoerythrin (300 .mu.l of
2.times.MES Stain Buffer (manufactured by Affymetrix), 24 .mu.l of
50 mg/ml acetylated BSA (manufactured by Affymetrix), 6 .mu.l of 1
mg/ml StreptAvidin-Phycoerythr- in (manufactured by Affymetrix),
270 .mu.l of water (manufactured by Affymetrix)) according to a
Technical Mannual of Affymetrix.
[0308] 4. Data Analysis
[0309] Detection of the signal intensity of the microarray was
carried out using Gene Chip Analysis Basic System and analysis
software (GCOS; GeneChip Operating Software 1.0) according to a
Technical Mannual of Affymetrix. Normalization was carried out
using the All Probe Sets in GCOS to adjust a signal in comparison
analysis. The comparison files of gene expression which were
compared (1) zinc depleted condition to zinc replete condition, (2)
oxidative stress condition to zinc replete condition and (3) carbon
starvation condition to zinc replete condition were created using
GCOS, respectively. The genes whose expressions were increased by
more than 0.3 at signal log ratio only in above comparison (1) were
shown in Table 4.
[0310] Sc-1159-1_at, Sc-1161-1_at, Sc-5030-1_at, Sc-2123-1_at
correspond to Sc YGL258W, Sc YGL256W, Sc YOL154W, Sc YKL175W,
respectively. And it is known that these genes are
transcriptionally induced in zinc depleted condition (Higgins, V.
J. et al., Appl. Environ. Microbiol., 69: 7535-7540(2003)).
Lg-4216-1_s_at was designed to correspond to Non-Sc YKL175W whose
function was assigned zinc ion transporter activity. It is known
that zinc ion transporter is transcriptionally induced in zinc
depleted condition.
[0311] In conclusion, it is shown that the molecular markers that
are highly induced in zinc depleted condition can be identified
using the bottom fermenting yeast DNA microarray.
4 TABLE 4 (2) oxidative (1) zinc deplete/zinc replete stress/zinc
replete (3) /zinc replete annotation Signal Log Signal Signal Log
Gene probe set Ratio Detection Change Log Ratio Detection Change
Ratio Detection Change name Type Sc-1159-1_at 3.1 P I -0.6 A NC
-0.5 A NC YGL258W Sc Lg-570-1_at 1.2 P I 0.1 P NC -0.7 A D YNL254C
Non-Sc Sc-116-1_at 1.1 P I -1.1 P D -1.2 P D YGL256W Sc
Lg-3847-1_at 0.9 P I -0.6 P D -1.1 P D YGL256W Non-Sc Sc-2889-1_at
0.7 P I -0.5 P D -1.8 P D YNL254C Sc Lg-4216-3__at 0.6 P I -0.6 P D
-0.4 P D YKL175W Non-Sc Sc-5030-1_at 0.5 P I -3.6 P D -3.8 P D
YOL154W Sc Sc-1160-1_at 0.4 P I -1.1 P D - P D YOL257C Sc
Lg-1751-1_at 0.4 P I -1 P D -0.2 P D YLR209C Non-Sc Sc-3567-1_at
0.4 P I 0.2 P NC -0.4 P NC YPL148C Sc Lg-3161-1_at 0.4 P I -0.8 P D
-0.9 P D YMR020W Non-Sc Sc-3_1_x_at 0.4 P I 0.2 P NC -1.3 P D
YL150W Sc Lg-4390-2_x_at 0.4 P I 0.3 P NC -0.5 P NC YLR339C Non-Sc
Sc-47-1_at 0.4 P I 0.2 P NC -2.3 P D YLR435W Sc Lg-51-1_s_at 0.4 P
I 0.2 P NC -2.3 P D YR312W Non-Sc Lg-139-1_at 0.3 P I -0.3 P NC
-4.1 A D YBR104W Non-Sc Lg-47-1_at 0.3 P I 0.1 P NC -2 P D YDR161W
Non-Sc Sc-1412-1_at 0.3 P I 0 P NC -2 P D YGR08C Sc Lg-961-1_at 0.3
P I 0.2 P NC -1.5 P D YGR103W Non-Sc Sc-2122-1_at 0.3 P I -0.3 P NC
0.1 P NC YKL176C Sc Sc-2123-1_at 0.3 P I -0.5 P D -1.1 P D YKL175W
Sc Sc-2209-1_at 0.3 P I 0.2 P NC -1.7 P D YKL072W Sc Sc-2356-1_at
0.3 P I -0.1 P NC -2.4 P D YLR129W Sc Lg-1955-1_at 0.3 P I -0.1 P
NC 0 P NC YMR096W Non-Sc Sc-2890-1_at 0.3 P I -0.3 P NC -1.2 P D
YNL253W Sc Lg-2100-1_at 0.3 P I -0.8 P D -1.7 P D YNL217W Non-Sc
Lg-2258-1_at 0.3 P I 0.1 P NC -1.8 P D YOL125W Non-Sc Sc-3203-1_at
0.3 P I 0.1 P NC -0.8 P D YOL0C Sc Sc-3651-1__at 0.3 P I 0.1 P NC
-0.1 P NC YP044C Sc Lg-2648-1_at 0.3 P I 0.1 P NC -2.1 P D YP
Non-Sc Lg-3014-1_at 0.3 P I -0.7 P D -0.3 P NC YJL055W Non-Sc
Sc-4034-1_at 0.3 P I 0.1 P NC 0.3 P NC YDR017C Sc Lg-360-1_at 0.3 P
I 0.4 P NC -2.1 P D YDR087C Non-Sc Sc-4163-1_at 0.3 P I 0.1 P NC
-2.5 M D YDR Sc Sc-4365-1_at 0.3 P I 0.3 P NC -1.4 P D YGR145W Sc
Sc-4454-_at 0.3 P I 0.4 P NC -1.8 P D YR197W Sc Lg-4608-2_at 0.3 P
I -0.3 P D -1.9 P D YNL112W Non-Sc Lg-4622-1_at 0.3 P I 0.1 P NC
-2.5 P D YNL062C Non-Sc Sc-5321-1_at 0.3 P I 0.4 P NC -1.1 P D
YGR272C Sc Lg-5125-1_at 0.3 P I 0.2 P NC -2.4 P D YOR101C
Non-Sc
[0312] Signal Log Ratio (2.sup.n) indicates the magnitude and
direction of a transcript when two arrays are compared. Detection
indicates whether a transcript is reliably detected (P; Present) or
not detected (A: Absent) based on the Detection p-value calculated
by Detection Algorithm with default paramater in GCOS. Change
indicates whether a transcript is reliably increased (I; Increase)
or decreased (D; Decrease) or not changed (NC; No Change) based on
the. Change p-value calculated by Change Algorithm with default
paramater in GCOS. Gene name indicates where the corresponding
probe set was designed. Type indicates whether a gene is Sc ORF
(Sc) or Non-Sc ORF (Non-Sc).
Example 19
Gene Expression Analysis of Brewing Yeast Under Beer Fermenting
Condition
[0313] Fermentation test using strain 34/70 was carried out under
the following condition.
[0314] Original extract: 12.84%
[0315] Fermentation scale: 2 liters
[0316] Dissolved oxygen concentration: about 9 ppm
[0317] Fermentation temperature: 15.degree. C.
[0318] Pitching rate: 10 g of wet yeast cells/2 L of wort
[0319] Wort was periodically sampled and monitored in cell growth
(OD600) (FIG. 12-(a)) and apparent extract (FIG. 12-(b)). mRNA was
extracted from the cells withdrawn from the fermentation tubes 42
hours after inoculation, biotin labeled and hybridized to the
bottom fermenting yeast DNA microarray as described in example 18.
Detection of the signal intensity was carried out using a Gene Chip
Analysis Basic System and analysis soft ware (GCOS; GeneChip
Operating Software 1.0) manufactured by Affymetrix.
[0320] There were more than a few genes whose Sc type probe sets
and non-Sc type probe sets showed quite different signal
intensities. Examples of SSU1 genes and MET14 genes, which are
related to sulfite production during beer fermentation are shown in
Table 5. In the case of SSU1 genes and MET14 genes of strain 34/70,
the expressions of non-Sc type were higher than those of Sc type,
by 3.4-fold and 7-fold, respectively.
[0321] In order to confirm this difference is due to neither the
difference of hybridization efficiency of each probe set nor cross
hybridization between Sc and non-Sc type probe sets, comparative
genomic hybridization with the bottom fermenting yeast DNA
microarray was carried out using strain 34/70, a laboratory strain
(S. cerevisiae) S288C and S. carlsbergensis strain IF011023. The
preparation of genomic DNA, hybridization to DNA microarray and
detection of the signal intensities were carried out with the
method described before. As shown in Table 6, the ratio of signal
intensity of non-Sc type to that of Sc type was 1.0 for SSU1 genes
and 1.3 for MET14 genes in strain 34/70. This result shows that
hybridization efficiencies of Sc and non-Sc probe sets were almost
the same.
[0322] Furthermore, strain S288C, which doesn't have non-Sc type
genes, showed very low signal intensities to non-Sc type probe
sets, and strain IFO11023, which doesn't have Sc type SSU1 gene and
Sc type MET14, showed very low signal intensities to Sc type SSU1
and Sc type MET14 probe sets. These results clearly show that cross
hybridization did not occur between Sc and non-Sc type probe
sets.
[0323] From these results, in strain 34/70, the expressions of
non-Sc SSU1 and non-Sc MET14 were significantly higher than those
of Sc SSU1 and Sc MET14, respectively. These genes are thought to
be candidates which contribute to the high sulfite production
ability of bottom fermenting yeasts.
[0324] In conclusion, it was demonstrated that gene expression
analysis of brewing yeast strains using the bottom fermenting yeast
DNA microarray was useful for the selection of gene(s) for
functional analysis.
5 TABLE 5 gene Sc SSU1 non Sc SSU1 Sc MET14 non Sc MET14 probe set
Sc-3594- 1_at Lg-3333-1_at Sc-2246-1_at Lg-1564-1_at signal 145.2
490.4 177.3 1245.8 intensity
[0325]
6TABLE 6 gene Sc SSU1 non Sc SSU1 Sc MET14 non Sc MET14 strains
probe set Sc-3594-1_at Lg-3333-1_at Sc-2246-1_at Lg-1564-1_at 34/70
signal 360.9 356.8 244.2 324.8 S288C intensity 516.2 6.5 405.3 13.4
S. carlsbergensis IF011023 8.5 746.9 6.8 508.4
Example 20
Classification of Brewing Strains by Comparative Genomic
Hybridization with the Bottom Fermenting Yeast DNA Microarray
[0326] Preparation of yeast genomic DNA and hybridization to the
DNA microarray was carried out as described in (Example 8).
Detection of the signal intensity of microarray was carried out
using a Gene Chip Analysis Basic System and analysis soft ware
(GCOS; GeneChip Operating. Software 1.0) manufactured by
Affymetrix. The percentage of probes, to which the DNA of brewing
yeast hybridized was calculated and the identity between strain
34/70 and the tested strain was estimated as shown in Table 7.
Strains BH225, BH232 and BH235 hybridized to more than 99% of both
Sc type and non-Sc type probes of the the bottom fermenting yeast
DNA microarray. It suggests that these strains are very similar to
strain 34/70, and that this microarray is useful for the gene
expression analysis of these strains. On the other hand, strain
BH212 showed relatively low (97.8 and 97.7% for Sc type and non-Sc
type probe, respectively) percentage of hybridization, which means
this strain is a little bit different from strain 34/70. From these
results, relationship among lager brewing strains can be estimated
and classification of lager brewing strains can be carried out.
[0327] From the result of the analysis of strain BH212, some loci
which showed very low signal intensities were found. They may be
lost in strain BH212 or their sequences may be different from those
of strain 34/70. In contrast, some loci which showed high signal
intensities were also found. These may be high in copy number in
strain BH212. Such loci can be selected for functional analysis
because they may contribute to the difference of fermentation
characteristics between strain BH212 and strain 34/70.
7TABLE 7 percentage of hybridized probes strain No. 34/70 BH225
BH232 BH235 BH212 Sc type 99.6 99.8 99.8 99.8 97.8 non-Sc type 99.5
99.9 99.9 99.6 97.7
Example 21
Detection of Nucleotide Polymorphism
[0328] Furthermore, (single) nucleotide polymorphism was detectable
by the analysis of comparative genomic hybridization. The sets of
oligonucleotides for each probe consist of Perfect Match
oligonucleotide (PM) which is identical to the sequence of strain
34/70 and MisMatch oligonucleotide (MM) which contains a single
base mismatch in the central position of the oligonucleotide.
Genomic DNA of a laboratory strain S288C was hybridized to the
bottom fermenting yeast DNA microarray. As shown in Table 8, probes
which showed higher (more than 5-fold) signal in MM than in PM had
single nucleotide polymorphism.
8TABLE 8 signal of signal of MM probe PM probe probe DNA sequence
of PM GAATCAATTAACTTATGGTTTCTTA
.vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline.
.vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline. Mt-6s at
653 337 112.38 634.39 DNA sequence of GAATCAATTAACATATGGTTTCTTA
tested strain .vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline. DNA sequence
of MM GAATCAATTAACATATGGTTTCTTA
[0329] A database compiling the data of the whole genome sequences
of an industrial yeast or, particularly, of a brewing yeast used
for the production of alcoholic beverages such as beer is prepared.
Using such a database, genes of brewing yeast are selected, and
functions of the genes are analyzed by disruption or overexpression
in yeast cells, and find genes participating in the desired brewing
character. Furthermore, it is possible to breed yeast strains by
controlling the expression of the said genes, and produce an
alcohol or an alcoholic beverage where productivity and quality are
improved, such as alcoholic beverages with high concentration of
sulfite which shows antioxidant activity in the product, excellent
flavor stability and a longer quality preservation period.
[0330] Based on the database compiling the data of the whole genome
sequences of an industrial yeast or, particularly, of a brewing
yeast, a DNA array is produced. Using the DNA array, it is possible
to analyze functions of the genes, classify industrial yeasts and
detect nucleotide polymorphism and so on.
Sequence CWU 1
1
32 1 1377 DNA Saccharomyces sp. 1 atggtcgcta gttggatgct cactgccaca
agggatttca accctttcat gtttgtcatg 60 gttatggggg tcggtatttc
atcgaatatt ctgtacagct tcccgtatcc ggcgaggtgg 120 ctgaggatat
gctcgtacat catgtttgcc attacatgtt tgattttcat ctctgtacag 180
gcgctgcagc ttttgcacat ggtcatctat atcaaagaaa aaagctttag agattacttc
240 aatgaatatt tcagaagtct gaagtacaat ttattttggg gtacttatcc
catgggatta 300 gtaacaatca taaatttttt gggggcgctg tcacaaaaat
ttaccacgac aagccctgcg 360 aatgccaagc acttgatcat ttttgtttac
gtcctgtggt ggtatgacct cgcggtttgt 420 ttagtaaccg cttgggggat
ttcattcctc atctggcaaa agtactactt cgtggacggg 480 gttggaaatc
actcttcata cagttcacga atggcttccg accacatgaa aagcgtactg 540
ttgctagata tcattccgct ggtcgttgtc gcttcgagcg gtgggacatt tacaatgtca
600 aaaatattcg gtaccacttt tgataggaat attcaattgc taacactggt
catctgtgcc 660 ctggtttggc tacacgctct tatatttgtc tttattctga
ttacaatata cttctggaat 720 ctttacatca ataagatacc accaatgacg
caggtattta cgttgttctt ggtattgggg 780 ccattgggcc aaggaagttt
tggtattttg ttgcttactg acaatataag aaagtatgta 840 gaaaaatact
acccaaggga aaacatcacc atggaacaag aaatactaac cattatggtt 900
ccgtggtgtt tcaaggttct gggcatgaca tttgctttgg cattaatcgc tatgggttac
960 ttctttacgg taatttccct tatttcgatt ttatcatact acaatgaaag
agttgttgac 1020 aatgaaacag gcaaagtgaa aaggatctac actttccata
aaggtttctg ggggatgact 1080 ttcccgatgg gtaccatgtc tttgggaaac
gaggagctgt atctgcaata caaccagtat 1140 gttcccttat atgcattcag
agtcatagct accatatatg gtggtatttg tgtttgctgg 1200 tcaatcttat
gcctctcgtg cacgttgtat ggttacctga aaacgattct ccatgctgcc 1260
cgtaaacctt cgtttttatc agaggaaggg acggagaaga ctgtcaattc tcctttcaac
1320 agcatcgaaa gtgtggagga atcaaactcg gctatcgata gtacatattt aacataa
1377 2 609 DNA Saccharomyces sp. 2 atggctacta atatcacttg gcatccaaat
cttacctacg acgaacgtaa ggaattaaga 60 aagcaagacg gctgtaccgt
ttggttgacc ggtctaagtg cgtcaggaaa aagtacaata 120 gcttgtgcac
tggaacaatt actgcttcaa aaaaacttat ctgcttatag gttagatggt 180
gataacattc gttttggttt gaataaggat ttgggcttct cagaaaagga cagaaatgaa
240 aacattcgta gaattagtga agtatccaag ctattcgctg attcgtgtgc
tgtatccatc 300 acttcattta tttccccata cagagtcgat agagacagag
cccgtgattt acataaggaa 360 gcaggcttga agttcattga aatttttgtt
gatgttccat tagaagtcgc tgagcaaaga 420 gaccctaagg gtttgtataa
gaaagccaga gaaggtgtga ttaaagagtt cactggtatt 480 tcagctcctt
acgaagctcc aaaggcccca gagttgcatt taagaactga ccaaaagact 540
gttgaagaat gtgctgctat catttatgag tacctggtca atgagaagat tatccggaag
600 catctataa 609 3 458 PRT Saccharomyces sp. 3 Met Val Ala Ser Trp
Met Leu Thr Ala Thr Arg Asp Phe Asn Pro Phe 1 5 10 15 Met Phe Val
Met Val Met Gly Val Gly Ile Ser Ser Asn Ile Leu Tyr 20 25 30 Ser
Phe Pro Tyr Pro Ala Arg Trp Leu Arg Ile Cys Ser Thr Ile Met 35 40
45 Phe Ala Ile Thr Cys Leu Ile Phe Ile Ser Val Gln Ala Leu Gln Leu
50 55 60 Leu His Met Val Ile Tyr Ile Lys Glu Lys Ser Phe Arg Asp
Tyr Phe 65 70 75 80 Asn Glu Tyr Phe Arg Ser Leu Lys Tyr Asn Leu Phe
Trp Gly Thr Tyr 85 90 95 Pro Met Gly Leu Val Thr Ile Ile Asn Phe
Leu Gly Ala Leu Ser Gln 100 105 110 Lys Phe Thr Thr Thr Ser Pro Ala
Asn Ala Lys His Leu Ile Ile Phe 115 120 125 Val Tyr Val Leu Trp Trp
Tyr Asp Leu Ala Val Cys Leu Val Thr Ala 130 135 140 Trp Gly Ile Ser
Phe Leu Ile Trp Gln Lys Tyr Tyr Phe Val Asp Gly 145 150 155 160 Val
Gly Asn His Ser Ser Tyr Ser Ser Arg Met Ala Ser Asp His Met 165 170
175 Lys Ser Val Leu Leu Leu Asp Ile Ile Pro Leu Val Val Val Ala Ser
180 185 190 Ser Gly Gly Thr Phe Thr Met Ser Lys Ile Phe Gly Thr Thr
Phe Asp 195 200 205 Arg Asn Ile Gln Leu Leu Thr Leu Val Ile Cys Ala
Leu Val Trp Leu 210 215 220 His Ala Leu Ile Phe Val Phe Ile Leu Ile
Thr Ile Tyr Phe Trp Asn 225 230 235 240 Leu Tyr Ile Asn Lys Ile Pro
Pro Met Thr Gln Val Phe Thr Leu Phe 245 250 255 Leu Val Leu Gly Pro
Leu Gly Gln Gly Ser Phe Gly Ile Leu Leu Leu 260 265 270 Thr Asp Asn
Ile Arg Lys Tyr Val Glu Lys Tyr Tyr Pro Arg Glu Asn 275 280 285 Ile
Thr Met Glu Gln Glu Ile Leu Thr Ile Met Val Pro Trp Cys Phe 290 295
300 Lys Val Leu Gly Met Thr Phe Ala Leu Ala Leu Ile Ala Met Gly Tyr
305 310 315 320 Phe Phe Thr Val Ile Ser Leu Ile Ser Ile Leu Ser Tyr
Tyr Asn Glu 325 330 335 Arg Val Val Asp Asn Glu Thr Gly Lys Val Lys
Arg Ile Tyr Thr Phe 340 345 350 His Lys Gly Phe Trp Gly Met Thr Phe
Pro Met Gly Thr Met Ser Leu 355 360 365 Gly Asn Glu Glu Leu Tyr Leu
Gln Tyr Asn Gln Tyr Val Pro Leu Tyr 370 375 380 Ala Phe Arg Val Ile
Ala Thr Ile Tyr Gly Gly Ile Cys Val Cys Trp 385 390 395 400 Ser Ile
Leu Cys Leu Ser Cys Thr Leu Tyr Gly Tyr Leu Lys Thr Ile 405 410 415
Leu His Ala Ala Arg Lys Pro Ser Phe Leu Ser Glu Glu Gly Thr Glu 420
425 430 Lys Thr Val Asn Ser Pro Phe Asn Ser Ile Glu Ser Val Glu Glu
Ser 435 440 445 Asn Ser Ala Ile Asp Ser Thr Tyr Leu Thr 450 455 4
202 PRT Saccharomyces sp. 4 Met Ala Thr Asn Ile Thr Trp His Pro Asn
Leu Thr Tyr Asp Glu Arg 1 5 10 15 Lys Glu Leu Arg Lys Gln Asp Gly
Cys Thr Val Trp Leu Thr Gly Leu 20 25 30 Ser Ala Ser Gly Lys Ser
Thr Ile Ala Cys Ala Leu Glu Gln Leu Leu 35 40 45 Leu Gln Lys Asn
Leu Ser Ala Tyr Arg Leu Asp Gly Asp Asn Ile Arg 50 55 60 Phe Gly
Leu Asn Lys Asp Leu Gly Phe Ser Glu Lys Asp Arg Asn Glu 65 70 75 80
Asn Ile Arg Arg Ile Ser Glu Val Ser Lys Leu Phe Ala Asp Ser Cys 85
90 95 Ala Val Ser Ile Thr Ser Phe Ile Ser Pro Tyr Arg Val Asp Arg
Asp 100 105 110 Arg Ala Arg Asp Leu His Lys Glu Ala Gly Leu Lys Phe
Ile Glu Ile 115 120 125 Phe Val Asp Val Pro Leu Glu Val Ala Glu Gln
Arg Asp Pro Lys Gly 130 135 140 Leu Tyr Lys Lys Ala Arg Glu Gly Val
Ile Lys Glu Phe Thr Gly Ile 145 150 155 160 Ser Ala Pro Tyr Glu Ala
Pro Lys Ala Pro Glu Leu His Leu Arg Thr 165 170 175 Asp Gln Lys Thr
Val Glu Glu Cys Ala Ala Ile Ile Tyr Glu Tyr Leu 180 185 190 Val Asn
Glu Lys Ile Ile Arg Lys His Leu 195 200 5 15 DNA Artificial
Sequence M13_for 5 agtcacgacg ttgta 15 6 17 DNA Artificial Sequence
M13_rv 6 caggaaacag ctatgac 17 7 22 DNA Artificial Sequence
SS-cosF.1 7 aggcgtatca cgaggccctt tc 22 8 29 DNA Artificial
Sequence SS-cosF.1 8 cttatcgatg ataagcggtc aaacatgag 29 9 36 DNA
Artificial Sequence XVI-1(L)cer-95894 9 cgcaagctcc gtacgttcaa
cattcttatg aacggc 36 10 36 DNA Artificial Sequence
XVI-1(R)nonSc-106302rv 10 gcatcatcgt cgtgatcctt ctttggcaaa tgcagg
36 11 36 DNA Artificial Sequence XVI-2(L)cer-859737 11 gcgggtattt
tgatggtaaa tctacaagcc ctcggc 36 12 35 DNA Artificial Sequence
XVI-2(R)nonSc-864595rv 12 cccagacaca gtttccagta tcatcctcgc agaac 35
13 26 DNA Artificial Sequence SacI-nonScSSU1_for1 13 gagctcatgg
tcgctagttg gatgct 26 14 26 DNA Artificial Sequence
BgIII-nonScSSU1_rv1460 14 agatctcagc ttcagcccaa tccatt 26 15 26 DNA
Artificial Sequence SacI-ScSSU1_for1 15 gagctcatgg ttgccaattg
ggtact 26 16 26 DNA Artificial Sequence Bg1II-ScSSU1_rv1406 16
agatctctcc tacatgaaat gcttgc 26 17 120 DNA Artificial Sequence
nonScSSU1_for 17 atggtcgcta gttggatgct cactgccaca agggatttca
accctttcat atcgaatatt 60 ctgtacagct gtttgtcatg gttatggggg
tcggtatttc ccttgacagt cttgacgtgc 120 18 120 DNA Artificial Sequence
nonScSSU1_rv 18 tgttaaatat gtactatcga tagccgagtt tgattcctcc
acactttcga acagtcttct 60 ccgtcccttc ctctgataaa tgctgttgaa
aggagaattg cgcacttaac ttcgcatctg 120 19 120 DNA Artificial Sequence
SScSSU1_for 19 atggttgcca attgggtact tgctcttacg aggcagtttg
accccttcat gtttatgatg 60 gtcatgggtg tcggcatttc atcgaatatt
ctatatagct ccttgacagt cttgacgtgc 120 20 120 DNA Artificial Sequence
ScSSU1_rv 20 ttatgctaaa cgcgtaaaat ctagagccga gtttgattct tccacgcttt
caatgctgtt 60 atacggagaa actgtcgtct tttccgtacc tgactctgaa
cgcacttaac ttcgcatctg 120 21 120 DNA Artificial Sequence
nonScSSU1_for+pGAPAUR 21 atggtcgcta gttggatgct cactgccaca
agggatttca accctttcat gtttgtcatg 60 gttatggggg tcggtatttc
atcgaatatt ctgtacagct ccggagctta ccagttctca 120 22 120 DNA
Artificial Sequence nonScSSU1_rv+AUR1-C 22 tgttaaatat gtactatcga
tagccgagtt tgattcctcc acactttcga tgctgttgaa 60 aggagaattg
acagtcttct ccgtcccttc ctctgataaa tcgactctag aggatccaga 120 23 20
DNA Artificial Sequence ScSSU1_for331 23 tcgaaagcga acacgacgaa 20
24 21 DNA Artificial Sequence ScSSu1_982rv 24 cgacagaaat cacggtgaaa
a 21 25 22 DNA Artificial Sequence nonScSSU1_329 25 tgtcacaaaa
atttaccacg ac 22 26 22 DNA Artificial Sequence nonScSSU1_981rv 26
aagggaaatt accgtaaaga ag 22 27 21 DNA Artificial Sequence PDA1_for1
27 atgtttgtcg cacctgtatc t 21 28 18 DNA Artificial Sequence
PDA1_730rv 28 gattagaggc accatcac 18 29 33 DNA Artificial Sequence
SacI-nonSc-MET12_for-21 29 ctcgagctct cgtgaaattc attgaaacaa atg 33
30 30 DNA Artificial Sequence BamHI-nonSc-MET14_rv618 30 ggatccttat
aagatttata gatgcttccg 30 31 33 DNA Artificial Sequence
SacI-ScMET14_for 31 ctcgagctca gaaaagttgg aattatttct cca 33 32 30
DNA Artificial Sequence BamHI-ScMET14_rv 32 ggatccaatg tacagtaatc
ggtcaaatta 30
* * * * *
References