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 polyhydroxyalkanoates 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 from L-valine, as a carbon source, in comparison with its wild type, wherein more 3-hydroxyisobutyric acid is formed from 3-hydroxyisobutyryl-coenzyme A as a precursor, wherein activities of enzymes E 8 , E 60 , E 61 , E 79 , and E 80  are increased in the cell compared to its wild type, wherein:
 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 a 2-methylacyl-coenzyme A dehydrogenase EC 1.3.99.12   E 79  is a 2-oxoisovalerate dehydrogenase EC 1.2.4.4, and   E 80  is an amino acid transferase EC 2.6.1.42,   wherein the cell is an  Escherichia coli  cell.   
     
     
         2 . A method of preparing a genetically modified cell which is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid using 3-hydroxybutyryl-coenzyme A, as a precursors, the method comprising increasing, in the cell, the activity of the enzymes of  claim 1 , wherein the cell is an  Escherichia coli  cell. 
     
     
         3 . A genetically modified cell obtainable by the method of  claim 2 . 
     
     
         4 . A method of producing 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, the method comprising bringing a cell of  claim 1  into contact with a nutrient medium comprising L-valine, as a carbon source, under conditions under which 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid are formed from the carbon source, and, optionally, purifying the 3-hydroxyisobutyric acid from the nutrient medium. 
     
     
         5 . A method of preparing methacrylic acid or methacrylic ester, the method comprising:
 IA) preparing 3-hydroxyisobutyric acid by the method of  claim 4  and, optionally, neutralizing the 3-hydroxyisobutyric acid, and   IB) dehydrating the 3-hydroxyisobutyric acid with formation of methacrylic acid and, optionally, esterifying the methacrylic acid.   
     
     
         6 . A method of preparing methacrylic acid or methacrylic esters, the method comprising:
 IIA) preparing a polyhydroxyalkanoate based on 3-hydroxybutyric acid by the method of  claim 4 ,   IIB) cleaving the polyhydroxyalkanoates with formation of 3-hydroxyisobutyric acid and, optionally, neutralizing the 3-hydroxyisobutyric acid, and   IIC) dehydrating the 3-hydroxyisobutyric acid with formation of methacrylic acid and, optionally, esterifying the methacrylic acid.   
     
     
         7 . A method of preparing polymethacrylic acid or polymethacrylic esters, the method comprising:
 IIIA) preparing methacrylic acid by the method as claimed in  claim 5 , and   IIIB) free-radical polymerizing the methacrylic acid.   
     
     
         8 . A method of preparing polymethacrylic acid or polymethacrylic esters, the method comprising:
 IIIA) preparing methacrylic acid by the method as claimed in  claim 6 , and   IIIB) free-radical polymerizing the methacrylic acid.