Specification
TECHNICAL FIELD
The present invention relates to a cysteine synthase gene and to uses of the gene. The invention relates in particular to a brewer's yeast which produces alcoholic beverages of excellent flavor, alcoholic beverages produced using such a yeast, and a method of producing such alcoholic beverages. More specifically, the invention relates to YGR012W gene which codes for the cysteine synthase Ygr012wp in brewer's yeast, particularly to a yeast which improves the flavor of product by increasing the level of expression of the non-ScYGRD12W gene characteristic to beer yeast or ScYGR012W gene and to a method of producing alcoholic beverages using such a yeast.
BACKGROUND ART
The beer yeast used in the production of commercial Pusner-type light-colored beers has the property of filming hydrogen sulfide during the primary fermentation step. This hydrogen sulfide is one cause of the immature beer aroma that is undesirable for beer quality. To reduce this aroma below a threshold level, extension of secondary fermentation, period or extension of maturation period is carried out.
Research on the factors affecting the formation of hydrogen sulfide, (Jangaard, N.O., Gress, H.S. and Coe, R.W.: Amer. Soc. Brew. Chem. Proa, p. 46 (1973); Ruroiwa, Y. and Hashimoto, N.: Brew. Dig., 45,44 (1970); Hysert, D.W. and Morrison, KM.: J. Amer. Soc. Brew. Chem., 34, 25 (1976)), and research on the development of a low hydrogen sulfide producing yeast using a mutation process or a cell fusion process (Molzahrn, S.W.: J. Amer. Soc. Brew. Chem., 35, 54 (1977)) for towering the hydrogen sulfide level in beer have been reported.
All of these methods not only reduces the amount of hydrogen sulfide produced by yeast but also affects the other brewing properties of the yeast (fermentation rate, beer flavor). Hence, such a yeast that is well-suited for brewing beer has not been achieved yet. Recently, development of brewer's yeasts using genetic engineering technology has been carried out. Japanese Patent Application Laid-open No. H5-244955 discloses mat a beer yeast in which a DNA fragment coding for cystathionine ^synthase has been inserted reduces -the production of hydrogen sulfide. However, the degree of reduction was smalL That is to say, the amount of hydrogen sulfide produced by the transformant was about 60 to 80% of that produced by the parent strain.
m yeast metabolism, hydrogen sulfide is produced in the process of reducing sulfate ions (SO42-) taken up from the medjum. This metabolic system is a pathway for the bbsvnmesis of suhlr-cxjriaming
The present inventors made exhaustive studies to solve the above problems, and as a result succeeded in identifying and isolating a gene encoding a cysteine synthase from lager brewing yeast
10
Moreover, a yeast.in which the obtained gene was transformed and expressed was produced confirm reduction of the amount of hydrogen sulfide, production, thereby completing the present invention.
Thus, the present invention relates to a novel cysteine synthase gene existing in a la^er brewing yeast, to a protein encoded by said gene, to a transformed yeast in which the expression of said gene is controlled, to a method for controlling the amount of hydrogen sulfide production in a product by using a yeast in which the expression of said gene is controlled More specifically, the present invention provides the following polynucleotides, a vector comprising said polynucleotide, a transformed yeast introduced with said vector, a method for producing alcoholic beverages by using said transformed yeast, and the like.
(1) A polynucleotide selected from the group consisting of
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide comprising a porynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having a cysteine synthase, activity,
(d) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO:2, and having a cysteine synthase activity; S ' ^ "■r'
(e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:l under stringent conditions, and which encodes a protein having a cysteine synthase activity; and
(f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the protein of the amino acid sequence of SEQ ID N02 under stringent conditions, and which encodes a protein having a cysteine synthase activity.
(2) The porynucleotide of (1) above selected from the group consisting of
(g) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID
NO: 2, or encoding an amino acid sequence of SEQ ID NO: 2 wherein 1 to 10 amino acids thereof is
deleted, substituted, inserted, and/or added, and wherein said protein has a cysteine synthase activity,
(h) a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2, and having a cysteine synthase activity, and
(i) a polynucleotide which hybridizes to SEQ ID NO: 1 or which hybridizes to a
nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under high stringent conditions, and which encodes a protein having a cysteine synthase activity.
(3)' The polynucleotide of (1) above comprising a polynucleotide consisting of SEQ ID NO: 1.
(4) The polynucleotide of (1) above comprising a polynucleotide encoding a protein consisting of SEQ ID NO: 2.
(5) The polynucleotide of any one of (1) to (4) above, wherein the polynucleotide is DNA.
(6) A protein encoded by the polynucleotide of any one of (1) to (5) above.
(7) A vector comprising the polynucleotide of any one of (1) to (5) above.
(7a) The vector of (7) above, which comprises the expression cassette comprising the following components:
(x) a promoter that can be transcribed in a yeast cell;
(y) any of the polynucleotides described in (1) to (5) above linked to the promoter in a sense or antisense direction; and
(z) a signal that can function in a yeast with respect to transcription termination and polyadenyiation of a RNA molecule.
(8) A vector comprising the polynucleotide selected from the group consisting of
0) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6, or encoding an amino acid sequence of SEQ ID NO 6 wherein 1 to 10 amino acids thereof is deleted, substituted, inserted, and/or added, and wherein saMprotem has a cysteine synmase activity^
(k) a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 6, and having a cysteine synthase activity; and
(1) a polynucleotide which hybridizes to SEQ ID NO: 5 or which hybridizes to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 under high stringent conditions, and which encodes a protein having a cysteine synthase activity.
(9) A yeast, wherein the vector of (7) or (8) above is introduced.
(10) The yeast of (9) above, wherein hydrogen snffide-producing ability is reduced by
introducing the vector of (7) or (8) above.
(U)Theyea^of(10)above,wbfremahyi^gmsum^k^ioduc^ ability is reduced by increasing an expression level of the protein of (6) above.
(12) A method for producing an alcoholic beverage by using the yeast of any one of (9) to (11) above.
(13) The method for producing an alcoholic beverage of (12) above, wherein the brew is a malt beverage.
(14) The method fcr producing an alcoholic beverage of (12) above, wherein the brew is a
wine.
(15) An alcoholic beverage, which is produced by the method of any one of (12) to (14) above.
(16) A method for assessing a test yeast for its hydrogen sulfide-producing ability, comprising using a primer or a probe designed based on a nucleotide sequence of a cysteine synthase gene having the nucleotide sequence of SEQ ID NO; I or SEQ ID NO: 5.
(16a) A method for selecting a yeast having a low hydrogen sulfide-producing ability by using the method in (16) above.
(16b) A method for producing an alcoholic beverage (for example, beer) by using the yeast selected with the method in (16a) above.
(17) A method for assessing a test yeast for its hydrogen sulfide-producing capability,
comprising: culturing a test yeast; and measuring an expression level of a cysteine synthase gene
having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5.
(17a) A method for selecting a yeast having a tow hydrogen sulfide-producing capability, which comprises assessing a test yeast by the method described in (17) above and selecting a yeast having a high expression level of the cysteine synthase gene.
(17b) A method for producing an alcoholic beverage (for example, beer) by using the yeast selected with the method in (17a) above.
(18) A method for selecting a yeast, comprising: culturing test yeasts; quantifying the
protein of (6) above or measuring an expression level of a cysteine synthase gene having the
nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5; and selecting a test yeast having said
protein amount or said gene expression level according to a target capability of producing hydrogen
sulfide.
(18a) A method for selecting a yeast, comprising: culturing tes yeasts; measuring a hydrogen sulfide-producing capability or a cysteine synthase activity; and selecting a test yeast having a target capability of producing hydrogen sulfide or a target cysteine synthase activity.
(19) The method for selecting a yeast of (18) above, comprising: culturing a reference yeast and test yeasts; measuring an expression level of a cysteine synthase gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5 in each yeast; and selecting a test yeast having the gene expressed higher than that in the reference yeast
(20) The method for selecting a yeast of (18) above comprising: culturing a reference yeast and test yeasts; quantifying the protein of (6) above in each yeast; and selecting a test yeast having said protein for a larger amount than that in the reference yeast That is, the method for selecting a yeast of (18) above comprising: culturing plural yeasts; quantifying the protein of (6) above in each yeast; and selecting a test yeast having a large amount of the protein from them.
(21) A method for producing an alcoholic beverage comprising: conducting fermentation for producing an alcoholic beverage using the ysast according to any one of (9) to (11) or a yeast selected by the method according to any one of (18) to (20); and adjusting the production amount of hydrogen sulfide.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows the cell growth with time upon beer brewing testing. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 2 shows the extract consumption with time upon test brew of beer. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).
Figure 3 shows the expression behavior of non-ScYGR012W gene in yeasts upon test brew of beer. The horizontal axis represents fermentation time while the vertical axis represents the intensity of detected signal.
Figure 4 shows the cell growth with time upon test brew of beer using parent strain and non-ScYGR012W highly expressed strain. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 5 shows the sugar consumption with time upon test brew of beer using parent strain and non-ScYGR012W highly expressed strain. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).
Figure 6 shows the cell growth with time upon test brew of beer. The horizontal axis xcpresents fermentation time while the vertical axis represents optical density gt 660 nm (OD660)
Figure 7 shows the extract consurhption with time upon test brew of beer. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).
Figure 8 shows the expression behavior of ScYGR012W gene in yeasts upon test brew of beer. The horizontal axis represents fermentation time while the vertical axis represents the intensity of detected signal
Figure 9 shows the cell growth with time upon test brew of beer using parent strain and ScYGR012W highly expressed strain. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 10 shows the extract consumption with time upon test brew of beer using parent strain and ScYGR012W highly expressed strain. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).
BEST MODES FOR CARRYING OUT THE INVENTION
The present inveniors conceived that it is possible to lower hydrogen sulfide effectively by increasing a cysteine synthase activity of the yeast The present inventors have studied based on this conception and as a result, isolated and identified non-ScYGR012W gene encoding a cysteine synthase unique to lager brewing yeast based on the lager brewing yeast genome information mapped according to the method disclosed in Japanese Patent Application Laid-Open No. 2004-283169. The nucleotide sequence of the gene is represented by SEQ ID NO: 1. Further, an amino acid sequence of a protein encoded by the gene is represented by SEQ ID NO: 2. In addition, the present inventors have isolated and identified ScYGRO 12W gene encoding a cysteine synthase of lager brewing yeast The nucleotide sequence of the gene is represented by SEQ ID NO: 5. Further, an amino acid sequence of a protein encoded by the gene is represented by SEQ ID NO: 6.
1. Polynucleotide of the invention
First of all, the present invention provides (a) a polynucleotide comprising a polynucleotide of the nucleotide sequence of SEQ ID NO:l or SEQ ID NO: 5; and(b) a polynucleotide comprising a polynucleotide encoding a protein of the amino acid sequence of SEQ ED NO:2 or SEQ ID NO: 6. The polynucleotide can be DNA or RNA.
The target polynucleotide of the present invention is not limited to the polynucleotide encoding a cysteine synthase gene derived from lager brewing, yeast. and may include other polynucleotides encoding proteins having equivalent functions to said protein. Proteins with equivalent functions include, for exarnple, (c) a protein of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 with one or more amino acids thereof being deleted, substituted, inserted and/or added and having cysteine synthase activity.
Such proteins include a protein consisting of an amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 6 with, for example, 1 to 100,1 to 90,1 to 80,1 to 70,1 to 60,1 to 50,1 to 40,1 to 39, lto38, lto37, Ito36,lto35, Ito34,lto33,lto32,lto31,lto30, lto29, lto28, lto27,lto 26,1 to 25,1 to 24,1 to23,1 to 22,1 to 21,1 to 20,1 to 19,1 to 18,1 to 17,1 to 16,1 to 15,1 to 14, 1 to 13,1 to 12,1 to 11,1 to 10,1 to 9,1 to 8,1 to 7,1 to 6 (1 to several amino acids), 1 to 5,1 to 4,1 to 3, 1 to 2, or 1 amino acid residues thereof being deleted, substituted, inserted and/or added and having a cysteine synthase activity. In general, the number of deletions, substitutions, insertions, andVor additions is preferably smaller. In addition, such proteins include (d) a protein having an amino acid sequence with about 60% or higher, about 70% or higher, 71 % or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher,
86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99,5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher identity wife the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, and having a cysteine synthase activity. In general, the percentage identity is preferably higher.
Cysteine synthase activity may be measured, for example, by a method of Thomas et al. as described in J. Biol, Chem. 45: 28187-28192 (1994).
Furthermore, the present invention also contemplates (e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5 under stringent conditions and which encodes a protein having cysteine synthase activity; and (f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide complementary to a nucleotide sequence of encoding a protein of SEQ ID NO: 2 or SEQ ID NO: 6 under stringent conditions, and which encodes a protein having cysteine synthase activity.
Herein, "a polynucleotide that hybridizes under stringent conditions" refers to a polynucleotide, such as a DNA, obtained by a cobny hybridization technique, a plaque hybridization technique, a southern hybridization technique or the like using all or part of polynucleotide of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5, or polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 as a probe. The hybridization method may be a method described, for example, in MOLECULAR CLONING 3rd 'Ed., CURRENT PROTOCOLS IN MOISCXJIAR BIOLOGY John Wiley* Sons 1987-19^*7
The term "stringent conditions" as used herein may be any of bw stringency conditions, moderate stringency conditions or high stringency conditions. "Low stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 32°C. "Moderate stringency conditions" are, for example, 5 x SSC, .5 x Denhardt's solution, 0.5% SDS, 50% formamide at 42°C. "High stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 50°G Under these conditions, a polynucleotide, such as a DNA, with higher homofogy is expected to be obtained efficiently at higher temperature, although multiple factors are involved in hybridization stringency including temperature, probe concentration, probe length, ionic strength, time, salt concentration and others, and one skilled in the art may appropriately select these factors to realize similar stringency.
When a commercially available kit is used for hybridization, for example, Alkphos Direct Labeling Reagents (Amersham Pharmacia) may be used In this case, according to the attached protocol, after incubation with a labeled probe overnight, the membrane is washed with a primary
wash buffer containing 0.1% (w/v) SDS at 55°C, thereby detecting hybridized polynucleotide, such as DNA.
Other polynucleotides that can be hybridized include polynucleotides having about 60% or higher, about 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81 % or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 993%' or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher or 99.9% or higher identity to polynucleotides encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 as calculated by homology search software, such as FASTA and BLAST using de&ult parameters.
Identity between amino acid sequences or nucleotide sequences may be determined using algorithm BLAST by Karhn and Altschul (Proc Natl Acad. ScL USA, 87: 2264-2268,1990; Proc. Natl Acad ScL USA, 90:5873,1993). Programs called BLASTN and BLASTX based on BLAST algorithm have been developed (Altschul SF et aL, J. Mol Biol 215: 403, 1990). When a nucleotide sequence is sequenced using BLASTN, the parameters are, for example, score = 100 and word length = 12. When an amino acid sequence is sequenced using BLASTX, the parameters are, for example, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, default parameters for each of the programs are employed.
2, ProieiB of the present invention
The present invention also provides proteins encoded by any of the polynucleotides (a) to (1) above. A preferred protein of the present invention comprises an amino acid sequence of SEQ ID NO-.2 or SEQ ID NO: 6 with one or several amino acids thereof being deleted, substituted, inserted and/or added, and has a cysteine synthase activity.
Such protein includes those having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 with amino acid residues thereof of the number mentioned above being deleted, substituted, inserted and/or added and having a cysteine synthase activity. In addition, such protein includes those having homology as described above with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 and having a cysteine synthase activity.
Such proteins may be obtained by employing site-directed mutation described, for example, in MOLECULAR CLONING 3rd Ed, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Nuc. A cids. Res., 10: 6487 (1982), Proc Natl Acad ScL USA 79:6409 (1982X Gene 34:315 (1985), Nuc Acids. Res., 13: 4431 (1985), Proc. Nad. Acad. Sci. USA 82: 488 (1985).
Deletion, substitution, insertion and/or addition of one or more amino acid residues in an amino acid sequence of the protein of the invention means that one or more amino acid residues are deleted, substituted, inserted and/or added at any one or more positions in the same amino acid sequence. Two or more types of deletion, substitution, insertion and/or addition may occur concurrently.
Hereinafter, examples of mutually substitutable amino acid residues are enumerated Amino acid residues in the same group are mutually substitutable, The groups are provided below.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: asparatic acid, glutamic acid, isoasparatic acid, isoglutamic acid, 2-arninoadipic acid, 2-aminosuberic acid; Group C: asparagine, ghitamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-
Documents
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| 1 |
395-chenp-2009 form-26 20-07-2009.pdf |
2009-07-20 |
| 1 |
395-CHENP-2009-AbandonedLetter.pdf |
2017-07-25 |
| 2 |
395-chenp-2009 correspondence others 20-07-2009.pdf |
2009-07-20 |
| 2 |
395-CHENP-2009-FER.pdf |
2017-01-09 |
| 3 |
Form13_Address of Applicant Change_05-11-2009.pdf |
2009-11-05 |
| 3 |
0395-chenp-2009 abstract.pdf |
2011-09-02 |
| 4 |
Correspondence by Agent_Form13_05-11-2009.pdf |
2009-11-05 |
| 4 |
0395-chenp-2009 claims.pdf |
2011-09-02 |
| 5 |
395-CHENP-2009 FORM-6 05-11-2009.pdf |
2009-11-05 |
| 5 |
0395-chenp-2009 correspondnece-others.pdf |
2011-09-02 |
| 6 |
395-CHENP-2009 FORM-13 05-11-2009.pdf |
2009-11-05 |
| 6 |
0395-chenp-2009 description(complete).pdf |
2011-09-02 |
| 7 |
395-chenp-2009(form-18).pdf |
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0395-chenp-2009 drawings.pdf |
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0395-chenp-2009 pct.pdf |
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0395-chenp-2009 form-3.pdf |
2011-09-02 |
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0395-chenp-2009 form-5.pdf |
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0395-chenp-2009 form-3.pdf |
2011-09-02 |
| 10 |
0395-chenp-2009 form-5.pdf |
2011-09-02 |
| 11 |
0395-chenp-2009 form-1.pdf |
2011-09-02 |
| 11 |
0395-chenp-2009 pct.pdf |
2011-09-02 |
| 12 |
0395-chenp-2009 drawings.pdf |
2011-09-02 |
| 12 |
395-chenp-2009(form-18).pdf |
2011-09-02 |
| 13 |
0395-chenp-2009 description(complete).pdf |
2011-09-02 |
| 13 |
395-CHENP-2009 FORM-13 05-11-2009.pdf |
2009-11-05 |
| 14 |
0395-chenp-2009 correspondnece-others.pdf |
2011-09-02 |
| 14 |
395-CHENP-2009 FORM-6 05-11-2009.pdf |
2009-11-05 |
| 15 |
0395-chenp-2009 claims.pdf |
2011-09-02 |
| 15 |
Correspondence by Agent_Form13_05-11-2009.pdf |
2009-11-05 |
| 16 |
0395-chenp-2009 abstract.pdf |
2011-09-02 |
| 16 |
Form13_Address of Applicant Change_05-11-2009.pdf |
2009-11-05 |
| 17 |
395-chenp-2009 correspondence others 20-07-2009.pdf |
2009-07-20 |
| 17 |
395-CHENP-2009-FER.pdf |
2017-01-09 |
| 18 |
395-CHENP-2009-AbandonedLetter.pdf |
2017-07-25 |
| 18 |
395-chenp-2009 form-26 20-07-2009.pdf |
2009-07-20 |
Search Strategy
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395SearchReport_27-12-2016.pdf |