MICROBIOLOGICAL PRODUCTION OF 3-HYDROXYISOBUTYRIC ACID

• US 20100068773A1
• Filed: 06/01/2007
• Published: 03/18/2010
• Est. Priority Date: 06/02/2006
• Status: Abandoned Application

• Current Assignee: Evonik Rã¶Hm GmbH (Rag-Stiftung)
• Original Assignee: Evonik Rã¶Hm GmbH (Rag-Stiftung)
• Inventors: Buchholz, Stefen, May, Alexander, Eggeling, Lothar, Siegert, Hermann, Poetter, Markus, Marx, Achim, Alber, Birgit, Fuchs, Georg
• Application Number:
  US12/303,161
• Publication Number:
  US 20100068773A1
• Time in Patent Office: Days
• US Class Current: 435/135
• CPC Class Codes: C08F 120/06 Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
• CPC Class Codes: C08F 120/10 Esters
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1096 transferring nitrogenous groups (2.6)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12P 7/52 Propionic acid; Butyric acids
• CPC Class Codes: C12P 7/625 Polyesters of hydroxy carboxylic acids
• CPC Class Codes: C12Y 102/04004 3-Methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) (1.2.4.4), i.e. branched-chain-alpha-ketoacid dehydrogenase
• CPC Class Codes: C12Y 103/99012 2-Methylacyl-CoA dehydrogenase (1.3.99.12)
• CPC Class Codes: C12Y 206/01042 Branched-chain-amino-acid transaminase (2.6.1.42)
• CPC Class Codes: C12Y 301/02004 3-Hydroxyisobutyryl-CoA hydrolase (3.1.2.4)
• CPC Class Codes: C12Y 402/01017 Enoyl-CoA hydratase (4.2.1.17), i.e. crotonase

Abstract

The present invention relates to a cell which has been modified in comparison with its wild type in such a way that it is capable of forming more, by comparison with its wild, 3-hydroxyisobutyric acid or poly-hydroxyalkanoates based on 3-hydroxyisobutyric acid via methylmalonate-semialdehyde or 3-hydroxybutyryl-coenzyme A as precursors. The invention also relates to a method of generating a genetically modified cell, to the genetically modified cell obtainable by these methods, to a method of producing 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, to a method of producing methacrylic acid or methacrylic esters, and to a method of producing polymethacrylic acid or polymethacrylic esters. The present invention furthermore relates to an isolated DNA, to a vector, to the use of this vector for transforming a cell, to a transformed cell, and to a polypeptide.

Claims

1. A cell which has been genetically modified in comparison with its wild type in such a way that it is capable of forming more 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid in comparison with its wild type.

2. The cell as claimed in claim 1, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via methylmalonate semialdehyde as precursor.

3. The cell as claimed in claim 2, where the cell is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via succinyl-coenzyme A as intermediate.

4. The cell as claimed in claim 3, where the cell features an activity of an enzyme E₁, which catalyzes the conversion of succinyl-coenzyme A into methylmalonyl-coenzyme A, which is increased in comparison with its wild type.

5. The cell as claimed in claim 4, where the enzyme E₁ is a methylmalonyl-coenzyme A mutase (EC 5.4.99.2).

6. The cell as claimed in claim 3, where the cell features an activity of at least one of the following enzymes E₂ to E₄ which is increased in comparison with its wild type:

of an enzyme E₂, which catalyzes the conversion of methylmalonyl-coenzyme A into methyl malonate;

of an enzyme E₃, which catalyzes the conversion of methyl malonate into methylmalonate semialdehyde;

of an enzyme E₄ which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyrate.

7. The cell as claimed in claim 6, where the enzyme

E₂ is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17),

E₃ is an aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase (EC 1.2.3.1) and

E₄ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).

8. The cell as claimed in claim 3, where the cell features an activity of at least one of the following enzymes E₄, E₅, E₄ and E₇ which is increased in comparison with its wild type:

of an enzyme E₆, which catalyzes the conversion of (R) methylmalonyl-coenzyme A into (S) methylmalonyl-coenzyme A;

of an enzyme E₇, which catalyzes the conversion of (S) methylmalonyl-coenzyme A into propionyl-coenzyme A;

of an enzyme E₅, which catalyzes the conversion of propionyl-coenzyme A into methylmalonate semialdehyde;

of an enzyme E₄, which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyric acid.

9. The cell as claimed in claim 8, where the enzyme

E₆ is a methylmalonyl-coenzyme A epimerase (EC 5.1.99.1)

E₇ is a methylmalonyl-coenzyme A decarboxylase (EC 4.1.1.41),

E₅ is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and

E₄ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).

10. The cell as claimed in claim 3, where the cell features an activity of at least one of the following enzymes E₄, E₅ and E₇ which is increased in comparison with its wild type:

of an enzyme E₇, which catalyzes the conversion of methylmalonyl-coenzyme A into propionyl-coenzyme A;

of an enzyme E₅, which catalyzes the conversion of propionyl-coenzyme A into methylmalonate semialdehyde;

of an enzyme E₄, which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyric acid.

11. The cell as claimed in claim 10, where the enzyme

E₇ is a methylmalonyl-coenzyme A decarboxylase (EC 4.1.1.41),

E₅ is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and

E₄ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).

12. The cell as claimed in claim 3, where the cell features an activity of at least one of the following enzymes E₂₈ and E₄₆ which is increased in comparison with its wild type:

of an enzyme E₄₆, which catalyzes the conversion of L-glutamate into 2-oxoglutarate;

of an enzyme E₂₈, which catalyzes the conversion of 2-octoglutarate into succinyl-coenzyme A.

13. The cell as claimed in claim 12, where the enzyme

E₄₆ is a glutamate synthase (EC 1.4.1.13 or EC 1.4.1.14), a glutamate dehydrogenase (EC 1.4.1.2, EC 1.4.1.3 or EC 1.4.1.4) or an aspartate transaminase (EC 2.6.1.1 or EC 2.6.1.2) and

E₂₈ is a 2-oxoglutarate synthase (EC 1.2.7.3).

14. The cell as claimed in claim 2, where the cell is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via propionyl-coenzyme A as intermediate.

15. The cell as claimed in claim 14, where the cell features an activity of at least one of the following enzymes E₄, E₅ and E₄₇ to E₅₂ which is increased in comparison with its wild type:

of an enzyme E₄₇, which catalyzes the conversion of acetyl-coenzyme A into malonyl-coenzyme A;

of an enzyme E₄₈, which catalyzes the conversion of malonyl-coenzyme A into malonate semialdehyde;

of an enzyme E₄₉, which catalyzes the conversion of malonate semialdehyde into 3-hydroxypropionate;

of an enzyme E₅₀, which catalyzes the conversion of 3-hydroxypropionate into 3-hydroxypropionyl-coenzyme A;

of an enzyme E₅₁, which catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into acryloyl-coenzyme A;

of an enzyme E₅₂, which catalyzes the conversion of acryloyl-coenzyme A into propionyl-coenzyme A;

of an enzyme E₅, which catalyzes the conversion of propionyl-coenzyme A into methylmalonate semialdehyde;

of an enzyme E₄, which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyrate.

16. The cell as claimed in claim 15, where the enzyme

E₄ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35),

E₅ is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27),

E₄₇ is a malonyl-coenzyme A decarboxylase (EC 4.1.1.9), a malonate coenzyme A transferase (EC 2.8.3.3), a methylmalonyl-coenzyme A carboxytransferase (EC 2.1.3.1) or an acetyl-coenzyme A carboxylase (EC 6.4.1.2),

E₄₈ is a malonate-semialdehyde dehydrogenase (EC 1.2.1.18),

E₄₉ is a 3-hydroxypropionate dehydrogenase (EC 1.1.1.59),

E₅₀ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4),

E₅₁ is an enoyl-coenzyme A hydratase (EC 4.2.1.17) and

E₅₂ is an acyl-coenzyme A dehydrogenase (EC 1.3.99.3).

17. The cell as claimed in claim 2, where the cell is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via acryloyl-coenzyme A as intermediate.

18. The cell as claimed in claim 17, where the cell features an activity of at least one of the following enzymes E₁₀ to E₁₂, E₅₆, E₇₂ and E₇₃ which is increased in comparison with its wild type:

of an enzyme E₇₂, which catalyzes the conversion of beta-alanine into beta-alanyl-coenzyme A,

of an enzyme E₇₃, which catalyzes the conversion of beta-alanyl-coenzyme A into acrylyl-coenzyme A,

of an enzyme E₅₆, which catalyzes the conversion of acrylyl-coenzyme A into methylmalonyl-coenzyme A,

of an enzyme E₁₀, which catalyzes the conversion of methylmalonyl-coenzyme A into methyl malonate;

of an enzyme E₁₁, which catalyzes the conversion of methyl malonate into methylmalonate semialdehyde;

of an enzyme E₁₂, which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyric acid.

19. The cell as claimed in claim 18, where the enzyme

E₇₂ is a coenzyme A transferase (EC 2.8.3.1) or coenzyme A synthetase, preferably a coenzyme A transferase,

E₇₃ is a beta-alanyl-coenzyme A ammonia-lyase (EC 4.3.1.6),

E₅₆ is a crotonyl-coenzyme A decarboxylase

E₁₀ is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17),

E₁₁ is an aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase (EC 1.2.3.1) and

E₁₂ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).

20. The cell as claimed in claim 1, where the formation of 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via 3-hydroxybutyryl-coenzyme A as precursor.

21. The cell as claimed in claim 20, where the cell is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via isobutyryl coenzyme A as intermediate.

22. The cell as claimed in claim 21, where the cell features an activity of at least one of the following enzymes E₇₆ to E₇₉, E₆₀, E₆₁ and E₈ which is increased in comparison with its wild type:

of an enzyme E₇₆, which catalyzes the conversion of pyruvate into 2-acetolactate;

of an enzyme E₇₇, which catalyzes the conversion of 2-acetolactate into 2,3-dihydroxyisovalerate;

of an enzyme E₇₈, which catalyzes the conversion of 2,3-dihydroxyisolvalerate into 2-oxoisovalerate;

of an enzyme E₇₉, which catalyzes the conversion of 2-oxoisovalerate into isobutyryl-coenzyme A;

of an enzyme E₆₀, which catalyzes the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme A;

of an enzyme E₆₁, which catalyzes the conversion of methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;

of an enzyme E₈, which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

23. The cell as claimed in claim 22, where the enzyme

E₈ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4),

E₇₆ is an acetolactate synthase (EC 2.2.1.6),

E₇₇ is a dihydroxyisovalerate dehydrogenase (EC 1.1.1.86),

E₇₈ is a 2,3-dihydroxyisovalerate dehydratase (EC 4.2.1.9),

E₇₉ is a 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4),

E₆₀ is an acyl-coenzyme A dehydrogenase (EC 1.3.99.3), a butyryl-coenzyme A dehydrogenase (EC 1.3.99.2) or a 2-methylacyl-coenzyme A dehydrogenase (EC 1.3.99.12), and

E₆₁ is an enoyl-coenzyme A hydratase (EC 4.2.1.17).

24. The cell as claimed in claim 21, where the cell features an activity of at least one of the following enzymes E₈, E₆₀ to E₆₁ and E₇₉ to E₈₀ which is increased in comparison with its wild type:

of an enzyme E₈₀, which catalyzes the conversion of L-valine into 2-oxoisovalerate;

of an enzyme E₇₉, which catalyzes the conversion of 2-oxoisovalerate into isobutyryl-coenzyme A;

of an enzyme E₆₀, which catalyzes the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme A;

of an enzyme E₆₁, which catalyzes the conversion of methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;

of an enzyme E₈, which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

25. The cell as claimed in claim 24, where the enzyme

E₈ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4),

E₆₀ is an enoyl-coenzyme A hydratase (EC 4.2.1.17),

E₆₁ is an acyl-coenzyme A dehydrogenase (EC 1.3.99.3), a butyryl-coenzyme A dehydrogenase (EC 1.3.99.2) or a 2-methylacyl-coenzyme A dehydrogenase (EC 1.3.99.12),

E₇₉ is a 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4), and

E₈₀ is an amino acid transferase (EC 2.6.1.42).

26. The cell as claimed in claim 20, where the cell is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via 3-hydroxybutyryl-coenzyme A as intermediate.

27. The cell as claimed in claim 26, where the cell an activity of at least one of the following enzymes E₈, E₅₃, E₅₄ and E₈₂ which is increased in comparison with its wild type:

of an enzyme E₅₃, which catalyzes the conversion of acetyl-coenzyme A into acetoacetyl-coenzyme A;

of an enzyme E₅₄, which catalyzes the conversion of acetoacetyl-coenzyme A into 3-hydroxybutyryl-coenzyme A;

of an enzyme E₈₁, which catalyzes the conversion of 3-hydroxybutyryl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;

of an enzyme E₈, which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

28. The cell as claimed in claim 27, where the enzyme

E₈ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4)

E₅₃ is a β

-kethothiolase (EC 2.3.1.9),

E₅₄ is an acetoacetyl-coenzyme A reductase (EC 1.1.1.36), and

E₈₂ is an isobutyryl-coenzyme mutase (EC 5.4.99.13).

29. A method of preparing a genetically modified cell which is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via methylmalonate semialdehyde or 3-hydroxybutyryl-coenzyme A, as precursors, comprising the method step of increasing, in the cell, the activity of at least one of the enzymes mentioned in claim 2.

31. A method of producing 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, comprising the method step of bringing a cell as claimed in claim 1 into contact with a nutrient medium comprising, as carbon source, carbohydrates, glycerol, carbon dioxide, methane, methanol, L-valine or L-glutamate under conditions under which 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid are formed from the carbon source, and, if appropriate, purification of the 3-hydroxyisobutyric acid from the nutrient medium.

32. A method of preparing methacrylic acid or methacrylic esters, comprising the method steps

IA) preparation of 3-hydroxyisobutyric acid by a method as claimed in claim 31 and, if appropriate, neutralization of the 3-hydroxyisobutyric acid,

IB) dehydration of the 3-hydroxyisobutyric acid with formation of methacrylic acid and, if appropriate, esterification methacrylic acid.

33. A method of preparing methacrylic acid or methacrylic esters, comprising the method steps

IIA) preparation of polyhydroxyalkanoates based on 3-hydroxybutyric acid by a method as claimed in claim 31,

IIB) cleavage of the polyhydroxyalkanoates based on 3-hydroxyisobutyric acid with formation of 3-hydroxyisobutyric acid and, if appropriate, neutralization of the 3-hydroxyisobutyric acid,

IIC) dehydration of the 3-hydroxyisobutyric acid with formation of methacrylic acid and, if appropriate, esterification of the methacrylic acid.

34. A method of preparing polymethacrylic acid or polymethacrylic esters, comprising the method steps

IIIA) preparation of methacrylic acid by a method as claimed in claim 32,

IIIB) free-radical polymerization of the methacrylic acid,

it being possible, if appropriate, to esterify at least in part the carboxyl groups of the methacrylic acid or the carboxylate group of the methacrylate before or after the free-radical polymerization reaction.

40. A method of preparing polymethacrylic acid or polymethacrylic esters, comprising the method steps

IIIA) preparation of methacrylic acid by a method as claimed in claim 33,

IIIB) free-radical polymerization of the methacrylic acid,

it being possible, if appropriate, to esterify at least in part the carboxyl groups of the methacrylic acid or the carboxylate group of the methacrylate before or after the free-radical polymerization reaction.

41. The cell as claimed in claim 22, where the cell features an activity of at least one of the following enzymes E₈, E₆₀ to E₆₁ and E₇₉ to E₈₀ which is increased in comparison with its wild type:

of an enzyme E₈₀, which catalyzes the conversion of L-valine into 2-oxoisovalerate;

of an enzyme E₇₉, which catalyzes the conversion of 2-oxoisovalerate into isobutyryl-coenzyme A;

of an enzyme E₆₀, which catalyzes the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme A;

of an enzyme E₆₁, which catalyzes the conversion of methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;

of an enzyme E₈, which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

30. A cell obtainable by a method as claimed in 29.

35. An isolated DNA, which is selected from the following sequences:

a) a sequence as shown in SEQ ID No 03,

b) an intron-free sequence which is derived from a sequence as specified in a) and which codes for the same protein or peptide as the sequence as shown in SEQ ID No 03,

c) a sequence which codes for a protein or peptide which comprises the amino acid sequence as shown in SEQ ID No 04,

d) a sequence with at least 80% identity with a sequence as specified in a) to c),

e) a sequence which hybridizes, or, taking into consideration the degeneration of the genetic code, would hybridize, with the counter strain of a sequence as specified in any of groups a) to d),

a derivative of a sequence as specified in any of groups a) to e), obtained by substitution, addition, inversion and/or deletion of one or more bases, and

g) a sequence which is complementary to a sequence as specified in any of groups a) to f).

36. A vector, comprising a DNA sequence as specified in any of groups a) to f), as defined in claim 35.

38. A transformed cell, obtainable by transformation with a vector as claimed in claim 36.

37. (canceled)

39. An isolated polypeptide which features the amino acid sequence with the SEQ ID No 04 or an amino acid sequence obtained when no more than 10 amino acids in SEQ ID No 04 are deleted, inserted, substituted or else added to the C and/or N terminus of the amino acid sequence with the SEQ ID No 04.

42. A cell obtained by the method as claimed in 29.