Microbiological production of 3-hydroxyisobutyric acid

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.

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 E 1 , 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 E 1  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 E 2  to E 4  which is increased in comparison with its wild type:
 of an enzyme E 2 , which catalyzes the conversion of methylmalonyl-coenzyme A into methyl malonate;   of an enzyme E 3 , which catalyzes the conversion of methyl malonate into methylmalonate semialdehyde;   of an enzyme E 4  which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyrate.   
     
     
         7 . The cell as claimed in  claim 6 , where the enzyme
 E 2  is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17),   E 3  is an aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase (EC 1.2.3.1) and   E 4  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 E 4 , E 5 , E 4  and E 7  which is increased in comparison with its wild type:
 of an enzyme E 6 , which catalyzes the conversion of (R) methylmalonyl-coenzyme A into (S) methylmalonyl-coenzyme A;   of an enzyme E 7 , which catalyzes the conversion of (S) methylmalonyl-coenzyme A into propionyl-coenzyme A;   of an enzyme E 5 , which catalyzes the conversion of propionyl-coenzyme A into methylmalonate semialdehyde;   of an enzyme E 4 , which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyric acid.   
     
     
         9 . The cell as claimed in  claim 8 , where the enzyme
 E 6  is a methylmalonyl-coenzyme A epimerase (EC 5.1.99.1)   E 7  is a methylmalonyl-coenzyme A decarboxylase (EC 4.1.1.41),   E 5  is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and   E 4  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 E 4 , E 5  and E 7  which is increased in comparison with its wild type:
 of an enzyme E 7 , which catalyzes the conversion of methylmalonyl-coenzyme A into propionyl-coenzyme A;   of an enzyme E 5 , which catalyzes the conversion of propionyl-coenzyme A into methylmalonate semialdehyde;   of an enzyme E 4 , which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyric acid.   
     
     
         11 . The cell as claimed in  claim 10 , where the enzyme
 E 7  is a methylmalonyl-coenzyme A decarboxylase (EC 4.1.1.41),   E 5  is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and   E 4  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 E 28  and E 46  which is increased in comparison with its wild type:
 of an enzyme E 46 , which catalyzes the conversion of L-glutamate into 2-oxoglutarate;   of an enzyme E 28 , which catalyzes the conversion of 2-octoglutarate into succinyl-coenzyme A.   
     
     
         13 . The cell as claimed in  claim 12 , where the enzyme
 E 46  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   E 28  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 E 4 , E 5  and E 47  to E 52  which is increased in comparison with its wild type:
 of an enzyme E 47 , which catalyzes the conversion of acetyl-coenzyme A into malonyl-coenzyme A;   of an enzyme E 48 , which catalyzes the conversion of malonyl-coenzyme A into malonate semialdehyde;   of an enzyme E 49 , which catalyzes the conversion of malonate semialdehyde into 3-hydroxypropionate;   of an enzyme E 50 , which catalyzes the conversion of 3-hydroxypropionate into 3-hydroxypropionyl-coenzyme A;   of an enzyme E 51 , which catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into acryloyl-coenzyme A;   of an enzyme E 52 , which catalyzes the conversion of acryloyl-coenzyme A into propionyl-coenzyme A;   of an enzyme E 5 , which catalyzes the conversion of propionyl-coenzyme A into methylmalonate semialdehyde;   of an enzyme E 4 , which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyrate.   
     
     
         16 . The cell as claimed in  claim 15 , where the enzyme
 E 4  is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35),   E 5  is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27),   E 47  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),   E 48  is a malonate-semialdehyde dehydrogenase (EC 1.2.1.18),   E 49  is a 3-hydroxypropionate dehydrogenase (EC 1.1.1.59),   E 50  is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4),   E 51  is an enoyl-coenzyme A hydratase (EC 4.2.1.17) and   E 52  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 E 10  to E 12 , E 56 , E 72  and E 73  which is increased in comparison with its wild type:
 of an enzyme E 72 , which catalyzes the conversion of beta-alanine into beta-alanyl-coenzyme A,   of an enzyme E 73 , which catalyzes the conversion of beta-alanyl-coenzyme A into acrylyl-coenzyme A,   of an enzyme E 56 , which catalyzes the conversion of acrylyl-coenzyme A into methylmalonyl-coenzyme A,   of an enzyme E 10 , which catalyzes the conversion of methylmalonyl-coenzyme A into methyl malonate;   of an enzyme E 11 , which catalyzes the conversion of methyl malonate into methylmalonate semialdehyde;   of an enzyme E 12 , which catalyzes the conversion of methylmalonate semialdehyde into 3-hydroxyisobutyric acid.   
     
     
         19 . The cell as claimed in  claim 18 , where the enzyme
 E 72  is a coenzyme A transferase (EC 2.8.3.1) or coenzyme A synthetase, preferably a coenzyme A transferase,   E 73  is a beta-alanyl-coenzyme A ammonia-lyase (EC 4.3.1.6),   E 56  is a crotonyl-coenzyme A decarboxylase   E 10  is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17),   E 11  is an aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase (EC 1.2.3.1) and   E 12  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 E 76  to E 79 , E 60 , E 61  and E 8  which is increased in comparison with its wild type:
 of an enzyme E 76 , which catalyzes the conversion of pyruvate into 2-acetolactate;   of an enzyme E 77 , which catalyzes the conversion of 2-acetolactate into 2,3-dihydroxyisovalerate;   of an enzyme E 78 , which catalyzes the conversion of 2,3-dihydroxyisolvalerate into 2-oxoisovalerate;   of an enzyme E 79 , which catalyzes the conversion of 2-oxoisovalerate into isobutyryl-coenzyme A;   of an enzyme E 60 , which catalyzes the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme A;   of an enzyme E 61 , which catalyzes the conversion of methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;   of an enzyme E 8 , which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.   
     
     
         23 . The cell as claimed in  claim 22 , where the enzyme
 E 8  is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4),   E 76  is an acetolactate synthase (EC 2.2.1.6),   E 77  is a dihydroxyisovalerate dehydrogenase (EC 1.1.1.86),   E 78  is a 2,3-dihydroxyisovalerate dehydratase (EC 4.2.1.9),   E 79  is a 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4),   E 60  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   E 61  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 E 8 , E 60  to E 61  and E 79  to E 80  which is increased in comparison with its wild type:
 of an enzyme E 80 , which catalyzes the conversion of L-valine into 2-oxoisovalerate;   of an enzyme E 79 , which catalyzes the conversion of 2-oxoisovalerate into isobutyryl-coenzyme A;   of an enzyme E 60 , which catalyzes the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme A;   of an enzyme E 61 , which catalyzes the conversion of methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;   of an enzyme E 8 , which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.   
     
     
         25 . The cell as claimed in  claim 24 , where the enzyme
 E 8  is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4),   E 60  is an enoyl-coenzyme A hydratase (EC 4.2.1.17),   E 61  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),   E 79  is a 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4), and   E 80  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 E 8 , E 53 , E 54  and E 82  which is increased in comparison with its wild type:
 of an enzyme E 53 , which catalyzes the conversion of acetyl-coenzyme A into acetoacetyl-coenzyme A;   of an enzyme E 54 , which catalyzes the conversion of acetoacetyl-coenzyme A into 3-hydroxybutyryl-coenzyme A;   of an enzyme E 81 , which catalyzes the conversion of 3-hydroxybutyryl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;   of an enzyme E 8 , which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.   
     
     
         28 . The cell as claimed in  claim 27 , where the enzyme
 E 8  is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4)   E 53  is a β-kethothiolase (EC 2.3.1.9),   E 54  is an acetoacetyl-coenzyme A reductase (EC 1.1.1.36), and   E 82  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 . 
     
     
         30 . A cell obtainable by a method as claimed in  29 . 
     
     
         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.   
     
     
         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 . 
     
     
         37 . (canceled) 
     
     
         38 . A transformed cell, obtainable by transformation with a vector as claimed in  claim 36 . 
     
     
         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. 
     
     
         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 E 8 , E 60  to E 61  and E 79  to E 80  which is increased in comparison with its wild type:
 of an enzyme E 80 , which catalyzes the conversion of L-valine into 2-oxoisovalerate;   of an enzyme E 79 , which catalyzes the conversion of 2-oxoisovalerate into isobutyryl-coenzyme A;   of an enzyme E 60 , which catalyzes the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme A;   of an enzyme E 61 , which catalyzes the conversion of methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;   of an enzyme E 8 , which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.   
     
     
         42 . A cell obtained by the method as claimed in  29 .