Microorganism strains for the production of 2.3-butanediol

1. A recombinant yeast having a reduced pyruvate decarboxylase activity, in the genome of which has been inserted:

one or more nucleic acids encoding an acetolactate synthase or ALS,

one or more nucleic acids encoding an acetolactate decarboxylase or ALD,

one or more nucleic acids encoding a butanediol dehydrogenase or BDH, and

two or more copies of nucleic acids encoding a NADH oxidase or NOXE,

wherein the nucleic acid(s) encoding the acetolactate synthase or ALS is/are nucleic acid(s) selected from the group consisting of sequences having at least 65% nucleic acid identity with the nucleic acid sequences SEQ ID NO: 1, 3 and 5,

wherein the nucleic acid(s) encoding the acetolactate decarboxylase or ALD is/are nucleic acid(s) selected from the group consisting of sequences having at least 36% nucleic acid identity with the nucleic acid sequences SEQ ID NO: 7, 9 and 11,

wherein the nucleic acid(s) encoding the butanediol dehydrogenase or BDH is/are nucleic acid(s) selected from the group consisting of sequences having at least 63% nucleic acid identity with the nucleic acid sequences SEQ ID NO: 13, 15, 17 and 19, and

wherein the nucleic acid(s) encoding the NADH oxidase or NOXE is/are nucleic acid(s) selected from the group consisting of sequences having at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NO: 21, 23, 25 and 27.

2. The recombinant yeast according to claim 1 , wherein the said recombinant yeast comprises one or more DNA constructs selected in a group comprising the following formulae:

(I) 5′-[Gene 1] x1 -3′ and 5′-[Gene 2] x2 -3′ and 5′-[Gene 3] x3 -3′ and 5′-[Gene 4] x4 -3′,

(II) 5′-[Gene 1] x1 -[Gene 2] x2 -[Gene 3] x3 -3′ and 5′-[Gene 4] x4 -3′,

(III) 5′-[Gene 1] x1 -[Gene 2] x2 -3′ and 5′-[Gene 3] x3 -[Gene 4] x4 -3′,

(IV) 5′-[Gene 1] x1 -[Gene 2] x2 -[Gene 3] x3 -[Gene 4] x4 -3′, and

a combination thereof,

wherein:

“Gene 1” means a nucleic acid selected from a group comprising ALS, ALD, BDH or NOXE;

“Gene 2” means a nucleic acid selected from a group comprising ALS, ALD, BDH or NOXE but different from gene 1;

“Gene 3” means a nucleic acid selected from a group comprising ALS, ALD, BDH or NOXE but different from genes 1 and 2;

“Gene 4” means a nucleic acid selected from a group comprising ALS, ALD, BDH or NOXE but different from genes 1 to 3;

“ALS” is a nucleic acid encoding an acetolactate synthase;

“ALD” is a nucleic acid encoding an acetolactate decarboxylase;

“BDH” is a nucleic acid encoding a butanediol dehydrogenase;

“NOXE” is a nucleic acid encoding a NADH oxidase;

each of “x1”, “x2”, “x3” and “x4”, one independently from the others, represents an integer ranging from 0 to 50, and

provided that said recombinant yeast comprises at least one nucleic acid encoding for each of ALS, ALD, BDH and NOXE.

3. The recombinant yeast according to claim 2 , wherein the said recombinant yeast comprises at least one DNA construct of formula (II), wherein “Gene 4” means a nucleic acid encoding NADH oxidase.

4. The recombinant yeast according to claim 2 , wherein the said recombinant yeast comprises at least one DNA construct(s) of formula (IIa), identical or different, wherein each formula (IIa) has the following formula:

(IIa) 5′-[(prom5) y1 -Gene 1-term5] x5 -[prom1-Gene 1-term 1] x1 -[prom2-Gene 2-term2] x2 -[prom3-Gene 3-(term3) z1 ] x3 -3′ and 5′-[(prom4) y2 -Gene 4-(term4) z2 ] x4 -3′

wherein:

Gene 1, Gene 2, Gene 3 and Gene 4 are such as defined in claim 2 , and “x1”, “x2”, “x3” and “x4” are such as defined in claim 2 ;

“x5” represents an integer equal to 0 or 1;

“y1”, “y2”, “z1” and “z2”, one independently from the others, represent an integer equal to 0 or 1;

when said recombinant yeast comprises at least two DNA constructs of formula (IIa), then “x1” to “x5”, “y1”, “y2”, “z1” and “z2” may be identical or different;

“prom 1” is a regulatory sequence which controls the expression of the sequence encoding the gene 1;

“prom 2” is a regulatory sequence which controls the expression of the sequence encoding the gene 2;

“prom 3” is a regulatory sequence which controls the expression of the sequence encoding the gene 3;

“prom 4” is a regulatory sequence which controls the expression of the sequence encoding the gene 4;

“prom5” is a regulatory sequence which controls the expression of Gene 1, said prom5 being identical or different from prom1;

“term 1” is a transcription terminator sequence that ends expression of the sequence encoding the gene 1;

“term2” is a transcription terminator sequence that ends expression of the sequence encoding the gene 2;

“term3” is a transcription terminator sequence that ends expression of the sequence encoding the gene 3;

“term4” is a transcription terminator sequence that ends expression of the sequence encoding the gene 4; and

“term5” is a transcription terminator sequence that ends expression of Gene 1, said term5 being identical or different from term 1.

5. The recombinant yeast according to claim 2 , wherein the said recombinant yeast comprises at least one DNA construct(s) of formula (IIb), identical or different, wherein each formula (IIb) has the following formula:

(IIb) 5′-[(prom5) y1 -ALS-term5] x5 -[prom1-ALS-term1] x1 -[prom2-ALD-term2] x2 -[prom3-BDH-(term3) z1 ] x3 -3′ and 5′-[(prom4) y2 -NOXE-(term4) z2 ] x4 -3′

wherein:

“x5” represents an integer equal to 0 or 1; and “y1”, “y2”, “z1” and “z2” one independently from the others, represent an integer equal to 0 or 1;

when said recombinant yeast comprises at least two DNA constructs of formula (III)), then “x1” to “x5”, “y1”, “y2”, “z1” and “z2” may be identical or different;

“prom 1” is a regulatory sequence which controls the expression of the sequence encoding the acetolactate synthase;

“prom 2” is a regulatory sequence which controls the expression of the sequence encoding the acetolactate decarboxylase;

“prom 3” is a regulatory sequence which controls the expression of the sequence encoding the butanediol dehydrogenase;

“prom 4” is a regulatory sequence which controls the expression of the sequence encoding the NADH oxidase;

“prom5” is a regulatory sequence which controls the expression of the sequence encoding the acetolactate synthase, said prom5 being identical or different from prom 1;

“term 1” is a transcription terminator sequence that ends expression of the sequence encoding the acetolactate synthase;

“term2” is a transcription terminator sequence that ends expression of the sequence encoding the acetolactate decarboxylase;

“term3” is a transcription terminator sequence that ends expression of the sequence encoding the butanediol dehydrogenase;

“term4” is a transcription terminator sequence that ends expression of the sequence encoding the NADH oxidase; and

“term5” is a transcription terminator sequence that ends expression of the sequence encoding the acetolactate synthase, said term5 being identical or different from term 1.

6. The recombinant yeast according to claim 2 , wherein the recombinant yeast comprises at least two DNA constructs of formula (II), (IIa) or (IIb), provided that all copies of NOXE's nucleic acid are located at a single of the at least two DNA constructs of formula (II), (IIa) or (IIb).

7. The recombinant yeast according to claim 2 , wherein the said recombinant yeast comprises at least two DNA constructs of the following formulae (IIc) and (IId):

(IIc) 5′-[(prom5) y1 -ALS-term5] x5 -[prom 1-ALS-term 1] x1 -[prom2-ALD-term2] x2 -[prom3-BDH-(term3) z1 ] x3 -3′ and 5′-[(prom4) y2 -NOXE-(term4) z2 ] x6 -3′; and

(IId) 5′-[(prom5) y1 -ALS-term5] x5 -[prom 1-ALS-term 1] x1 -[prom2-ALD-term2] x2 -[prom3-BDH-(term3) z1 ] x3 -3′ and 5′-[(prom4) y2 -NOXE-(term4) z2 ] x7 -3′;

wherein:

“prom 1” is a regulatory sequence which controls the expression of the sequence encoding the gene 1;

“prom 2” is a regulatory sequence which controls the expression of the sequence encoding the gene 2;

“prom 3” is a regulatory sequence which controls the expression of the sequence encoding the gene 3;

“prom 4” is a regulatory sequence which controls the expression of the sequence encoding the gene 4;

“prom5” is a regulatory sequence which controls the expression of Gene 1, said prom5 being identical or different from prom1;

“term 1” is a transcription terminator sequence that ends expression of the sequence encoding the gene 1;

“term2” is a transcription terminator sequence that ends expression of the sequence encoding the gene 2;

“term3” is a transcription terminator sequence that ends expression of the sequence encoding the gene 3;

“term4” is a transcription terminator sequence that ends expression of the sequence encoding the gene 4; and

“term5” is a transcription terminator sequence that ends expression of Gene I, said term5 being identical or different from term 1;

“x5” represents an integer equal to 0 or 1,

“y1”, “y2”, “z1” and “z2” one independently from the others, represent an integer equal to 0 or 1;

“x1” to “x3”, “x5”, “y1”, “y2”, “z1” and “z2” for each formulae (IIc) and (IId) being identical or different; and

“x6” and “x7” represent integers ranging from 0 to 50, provided that one among “x6” and “x7” represents 0.

8. The recombinant yeast according to claim 1 , wherein each of nucleic acids encoding acetolactate synthase, acetolactate decarboxylase, butanediol dehydrogenase, and NADH oxidase is under the control of a promoter, identical or different, said promoters consisting of a sequence of nucleic acid selected from the group consisting of sequences having at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NO: 29 to 39, 49 and 50.

9. The recombinant yeast according to claim 1 , wherein each of nucleic acids encoding acetolactate synthase, acetolactate decarboxylase, butanediol dehydrogenase, and NADH oxidase is under the control of a transcription terminator, identical or different, said transcription terminators consisting of a sequence of nucleic acid selected from the group consisting of sequences having at least 80% nucleic acid identity with the nucleic acid sequence of SEQ ID NO: 40 to 48.

10. The recombinant yeast according to claim 1 , wherein the pyruvate decarboxylase activity is reduced by disruption of at least one pdc gene.

11. A method for producing 2,3-butanediol (BDO), said method comprising the steps of:

(a) culturing a recombinant yeast such as defined in claim 1 in an appropriate culture medium; and

(b) recovering the 2,3-butanediol (BDO).

12. The method according to claim 11 , wherein the said culture medium comprises a carbon source.