Method for analyzing genes of industrial yeasts

• US 20060099612A1
• Filed: 09/02/2005
• Published: 05/11/2006
• Est. Priority Date: 09/02/2004
• Status: Abandoned Application

• Current Assignee: Suntory Holdings Limited (Kotobuki Realty Co., Ltd.)
• Original Assignee: Suntory Ltd (Kotobuki Realty Co., Ltd.)
• Inventors: Nakao, Yoshihiro, Kodama, Yukiko, Nakamura, Norihisa, Ashikari, Toshihiko, Fujimura, Tomoko
• Application Number:
  US11/217,529
• Publication Number:
  US 20060099612A1
• Time in Patent Office: Days
• US Class Current: 435/6.18
• CPC Class Codes: C07K 14/39 from yeasts
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Abstract

An object of the present invention is to provide a method for analyzing gene of an industrial yeast. The method of the present invention comprises (a) analyzing the genome sequence of the industrial yeast; and

• • (c-1) selecting 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, or
  • (c-2) selecting a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae.

Claims

1. A method for analyzing gene of an industrial yeast comprising

(a) analyzing nucleotide sequence of the genome sequence of the industrial yeast;

and

(c-1) selecting 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, or

(c-2) selecting a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae.

2. The analyzing method according to claim 1, comprising,

(b) comparing the genome sequence of the industrial yeast with the genome sequence of Saccharomyces cerevisiae after the step (a) and before the step (c-1) or (c-2).

3. The analyzing method according to claim 1, comprising

(d) carrying out functional analysis of the selected gene after the step (c-1) or (c-2).

4. The analyzing method according to claim 3, which comprises screening genes involved in increase of productivity and/or improvement in flavor in the production of an alcohol or an alcoholic beverage by the functional analysis of the step (d).

5. The analyzing method according to claim 3, wherein the functional analysis is performed by disruption of a gene or overexpression of a gene.

6. The analyzing method according to claim 1, wherein the industrial yeast is a brewing yeast.

7. The analyzing method according to claim 6, wherein the brewing yeast is a beer yeast.

8. The analyzing method according to claim 7, wherein the beer yeast is a bottom fermenting yeast.

9. 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, or consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, which is obtained by the analyzing method according to any one of claims 1-8.

10. A gene library comprising one or more of genes of the industrial yeast each of which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, which is obtained by the analyzing method according to any one of claim 1.

11. The gene library according to claim 10, wherein the gene of the industrial yeast is comprised in, consists of or has any nucleotide sequence selected from SEQ ID NOs:

33 to 6236, SEQ ID NOs;

166154 to 166181, SEQ ID NOs;

166490 to 167042 and SEQ ID NOs;

173125 to 174603.

30. The gene according to claim 9, characterized in that concentration of sulfite in a culture medium of an industrial yeast increases when the gene is expressed in the yeast.

33. A recombinant vector containing the gene of claim 9.

34. A transformant comprising the recombinant vector of claim 33.

35. The transformant according to claim 34, which is a yeast of genus Saccharomyces.

38. A method for producing an alcohol or an alcoholic beverage, comprising culturing the transformant of claim 34.

39. 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 of claim 9 is controlled.

40. The breeding method according to claim 39, wherein the yeast belongs to the genus Saccharomyces.

41. Yeast obtained by the breeding method of claim 39.

42. A method for producing an alcohol or an alcoholic beverage comprising culturing the yeast of claim 41.

43. An alcohol or an alcoholic beverage which is produced by the producing method of claim 42.

12. A DNA array comprising one or more of DNAs, wherein each DNA is selected from at least one group of (1) to (4):

(1) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(2) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(3) DNA having a nucleotide sequence of any of SEQ ID NOs;

166490 to 167042, or DNA having a nucleotide sequence of any of SEQ ID NOs;

167043 to 173124; and

(4) DNA having a nucleotide sequence of any of SEQ ID NOs;

173125 to 174603, or DNA having a nucleotide sequence of any of SEQ ID NOs;

174604 to 190810.

13. The DNA array according to claim 12 comprising DNAs wherein at least one DNA is selected from every group of (1) to (4).

14. The DNA array according to claim 12, wherein the DNAs of (1) is comprised in, consists of or has any nucleotide sequence selected from SEQ ID NOs:

33 to 6236, SEQ ID NOs;

166154 to 166181, SEQ ID NOs;

6237 to 75336 and SEQ ID NOs;

166182 to 166489.

15. The DNA array according to any one of claim 12, which further comprises, in addition to at least one group of DNAs selected from (1) to (4), DNAs of the following (5) and/or (6):

(5) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having identity of more than 97% to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences; and

(6) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having identity of more than 94% to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences, and optionally DNAs of the following (5′

) and/or (6′

);

(5′

) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (5), or a nucleotide sequence complementary to the above nucleotide sequence; and

(6′

) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (6), or a nucleotide sequence complementary to the above nucleotide sequence.

16. The DNA array according to claim 15, wherein the DNAs of (5) has a nucleotide sequence of any one of SEQ ID NOs:

75337 to 82784, or SEQ ID NOs;

82785 to 166153.

17. The DNA array according to any one of claim 12, which comprises, in addition to at least one group of DNAs selected from (1) to (4), DNAs selected from at least one of (7) to (10):

(7) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (1), or a nucleotide sequence complementary to the above nucleotide sequence;

(8) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (2), or a nucleotide sequence complementary to the above nucleotide sequence;

(9) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (3), or a nucleotide sequence complementary to the above nucleotide sequence; and

(10) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (4), or a nucleotide sequence complementary to the above nucleotide sequence.

18. The DNA array according to claim 17, which comprises, in addition to at least one group of DNAs selected from (1) to (4), DNAs selected from at least one of (7-1) to (10-1):

(7-1) DNA consisting of a nucleotide sequence which contains mismatch of 1 base to the DNA of (1), or a nucleotide sequence complementary to the above nucleotide sequence;

(8-1) DNA consisting of a nucleotide sequence which contains mismatch of 1 base to the DNA of (2), or a nucleotide sequence complementary to the above nucleotide sequence;

(9-1) DNA consisting of a nucleotide sequence which contains mismatch of 1 base to the DNA of (3), or a nucleotide sequence complementary to the above nucleotide sequence; and

(10-1) DNA consisting of a nucleotide sequence which contains mismatch of 1 base to the DNA of (4), or a nucleotide sequence complementary to the above nucleotide sequence.

20. The DNA array according to claim 12 or 19, wherein the DNAs are oligonucleotides consisting of 10-30 nucleotides.

21. A method for analyzing genes of an industrial yeast comprising hybridizing genes of the industrial yeast to the DNA array according to claim 12 or 19.

22. A method for classifying an industrial yeast comprising

(a) hybridizing genomic DNA prepared from the industrial yeast to the DNA array of claim 15 or 19;

and

(b) calculating hybridization ratio to any of (1) to (4), and hybridization ratio to (5) and/or (6).

23. The method for classification according to claim 22,

wherein the DNA array comprises at least one group of DNAs selected from (1) to (4), at least one group of DNAs selected from (7) to (10), DNAs of (5) and/or (6) and DNAs of (5′

) and/or (6′

), and

percentage of genes which hybridize to DNAs of (1) and/or (2), but not to DNAs of (7) and/or (8) is determined as hybridization ratio to any of (1) to (4), and

percentage of genes which hybridize to DNAs of (5) and/or (6), but not to DNAs of (5′

) and/or (6′

) is determined as hybridization ratio to any of (5) and/or (6).

24. A method for detecting nucleotide polymorphism in genome of an industrial yeast comprising

(a) hybridizing genomic DNA prepared from the industrial yeast to the DNA array of any one of claims 17 or 19;

and

(b) selecting a gene wherein hybridization intensity to the DNAs of any of (7) to (10) is higher than hybridization intensity to the DNAs of any of (1) to (4).

25. A method for screening a useful strain of industrial yeast, comprising

(a) hybridizing genomic DNA prepared from the industrial yeast strain to the DNA array of claim 12 or 19;

and

(b) determining the industrial yeast strain as useful when the strain contain 1 or more genes wherein hybridization intensity thereof is 1.5 times higher, or ⅔

or less compared to an average hybridization intensity to the DNAs of any of (1) to (4).

26. A method for screening a gene of an industrial yeast, comprising

(a) hybridizing genomic DNA prepared from the industrial yeast strain to the DNA array of claim 12 or 19;

and

(b) selecting a gene wherein hybridization intensity thereof is 1.5 times higher, or ⅔

or less compared to an average hybridization intensity to the DNAs of any of (1) to (4).

46. A recombinant vector containing the nucleic acid of claim 21.

19. A DNA array comprising:

I) at least one group of DNAs selected from at least one group of (1) to (4);

(1) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(2) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(3) DNA having a nucleotide sequence of any of SEQ ID NOs;

166490 to 167042, or DNA having a nucleotide sequence of any of SEQ ID NOs;

167043 to 173124; and

(4) DNA having a nucleotide sequence of any of SEQ ID NOs;

173125 to 174603, or DNA having a nucleotide sequence of any of SEQ ID NOs;

174604 to 190810;

II) at least one group of DNAs selected from at least one group of (7) to (10);

(7) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (1), or a nucleotide sequence complementary to the above nucleotide sequence;

(8) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (2), or a nucleotide sequence complementary to the above nucleotide sequence;

(9) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (3), or a nucleotide sequence complementary to the above nucleotide sequence; and

(10) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (4), or a nucleotide sequence complementary to the above nucleotide sequence;

III) DNAs of the following (5) and/or (6);

(5) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino said sequence having identity of more than 97% to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotide selected from the above nucleotide sequences; and

(6) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having identity of more than 94% to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences; and

IV) DNAs of the following (5′

) and/or (6′

);

(5′

) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (5), or a nucleotide sequence complementary to the above nucleotide sequence; and

(6′

) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (6), or a nucleotide sequence complementary to the above nucleotide sequence.

27. A method for screening a gene of an industrial yeast, comprising

(a-1) hybridizing genomic DNA, cDNA or cRNA prepared from the industrial yeast to a DNA array comprising one or more of DNAs, wherein each DNA is selected from at least one group of (1) to (4):

(1) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence or an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(2) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence of having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(3) DNA having a nucleotide sequence of any of SEQ ID Nos;

166490 to 167042, or DNA having a nucleotide sequence of any of SEQ ID NOs;

167043 to 173124; and

(4) DNA having a nucleotide sequence of any of SEQ ID NOs;

173125 to 174603, or DNA having a nucleotide sequence of any of SEQ ID NOs;

174604 to 190810;

(a-2) independently from the step (a-1), hybridizing genomic DNA, cDNA or cRNA prepared from another industrial yeast to the DNA array comprising one or more of DNAs, wherein each DNA is selected from at least one group of (1) to (4);

(1) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(2) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from pen reading frames which consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequences;

(3) DNA having a nucleotide sequence of any of SEQ ID NOs;

166490 to 167042, or DNA having a nucleotide sequence of any of SEQ ID NOs;

167043 to 173124; and

(4) DNA having a nucleotide sequence of any of SEQ ID NOs;

173125 to 174603, or DNA having a nucleotide sequence of any of SEQ ID NOs;

174604 to 190810; and

(b) selecting a gene wherein hybridization intensity thereof to the DNAs of any of (1) to (4) at the step (a-1) is significantly different from hybridization intensity thereof at the step (a-2).

28. A method for screening a gene of an industrial yeast, comprising

(a-1) hybridizing cDNA or cRNA prepared from the industrial yeast to a DNA array comprising one or more of DNAs, wherein each DNA is selected from at least one group of (1) to (4):

(1) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(2) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(3) DNA having a nucleotide sequence of any of SEQ ID NOs;

166490 to 167042, or DNA having a nucleotide sequence of any of SEQ ID NOs;

167043 to 173124; and

(4) DNA having a nucleotide sequence of any of SEQ ID NOs;

173125 to 174603, or DNA having a nucleotide sequence of any of SEQ ID NOs;

174604 to 190810;

(a-2) independently from the step (a-1), hybridizing cDNA or cRNA prepared from the industrial yeast to a DNA array comprising one or more of DNAs, wherein each DNA is selected from at least one group of (1) to (4);

(1) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(2) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;

(3) DNA having a nucleotide sequence of any of SEQ ID NOs;

166490 to 167042, or DNA having a nucleotide sequence of any of SEQ ID NOs;

167043 to 173124; and

(4) DNA having a nucleotide sequence of any of SEQ ID NOs;

173125 to 174603, or DNA having a nucleotide sequence of any of SEQ ID NOs;

174604 to 190810;

wherein the industrial yeast of (a-2) has been cultured in a different condition from the culture condition for the industrial yeast of (a-1); and

(b) selecting a gene wherein hybridization intensity thereof to the DNAs of any of (1) to (4) at the step (a-1) is significantly different from hybridization intensity thereof at the step (a-2).

29. A gene obtained by the screening method of claim 28.

44. The gene according to claim 29 characterized in that concentration of sulfite in a culture medium of an industrial yeast increases when the gene is expressed in the yeast.

45. A recombinant vector containing the gene of claim 29.

47. A breeding method of yeast which is suitable for the production of an alcohol or alcoholic beverage, characterized in that, expression of the gene of claim 29 is controlled.

31. A nucleic acid encoding a polypeptide of any one of the following i) and ii):

i) a polypeptide having the amino acid sequence represented by SEQ ID NO;

3, and

ii) a polypeptide having an amino acid sequence wherein one or more amino acid residue(s) is deleted from, substituted for and/or added to the amino acid sequence represented by SEQ ID NO;

3, and having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.

32. The nucleic acid according to claim 31, which is selected from the following a) and b):

(a) a nucleic acid having the nucleotide sequence represented by SEQ ID NO;

1; and

(b) a nucleic acid having a nucleotide sequence which hybridizes to the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO;

1 under a stringent condition, and encodes a polypeptide having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.

48. A breeding method of yeast which is suitable for the production of an alcohol or an alcoholic beverage, characterized in that, expression of the nuclec acid of claim 31 is controlled.

36. A polypeptide of any one of the following i) and ii):

i) a polypeptide having the amino acid sequence represented by SEQ ID NO;

3; and

ii) a polypaptide having an amino acid sequence wherein one or more amino acid residue(s) is deleted from, substituted for and/or added to the amino acid sequence represented by SEQ ID N;

3, and having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.

37. The polypeptide according to claim 36, which is encoded by a nucleic acid selected from the following a) and b):

(a) a nucleic acid having the nucleotide sequence represented by SEQ ID NO;

1; and

(b) a nucleic acid having a nucleotide sequence which hybridizes to the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID No.1 under a stringent condition, and encodes a polypeptide having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.

49. A method for analyzing gene of an industrial yeast comprising

(c-1) selecting 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, or

(c-2) selecting a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae.