Process for preparing methacrylic acid or methacrylic esters

A process for preparing a methacrylic acid or a methacrylic ester, comprising preparing a 3-hydroxyisobutyric acid by a process comprising:

• culturing a genetically modified cell which forms 3-hydroxyisobutyric acid, or polyhydroxy-alkanoate comprising 3-hydroxyisobutyric acid via a pathway employing succinyl-coenzyme A as an intermediate and methylmalonate semialdehyde as precursor in a nutrient medium comprising a carbon source under a condition wherein at least a portion of the carbon source is enzymatically converted to 3-hydroxyisobutyric acid or a polyhydroxy-alkanoate comprising 3-hydroxyisobutyric acid,
• wherein the carbon source is at least one source selected from the group consisting of a carbohydrate, glycerol, L-valine, and L-glutamate;
• wherein the genetically modified cell comprises:
• (1) NCg11470, NCg11471, and NCg11472 genes encoding an enzyme E1 which catalyses conversion of succinyl-coenzyme A to methylmalonyl coenzyme A;
• (2) a gene of S. tokodaii encoding malonyl-coenzyme A reductase that provides enzyme activities of enzymes E2 and E3, wherein the enzyme E2 catalyses conversion of methylmalonyl-coenzyme A to methylmalonate, and the enzyme E3 catalyses conversion of methylmalonate to methylmalonate semialdehyde;
• and
• (3) a gene of Thermus thermophilus encoding an enzyme E4 which catalyses conversion of methylmalonate semialdehyde to 3-hydroxyisobutyrate;
• wherein the genes (1), (2), and (3) express respective enzymes which catalyze the formation of 3-hydroxyisobutyric acid by the genetically modified cells when the cells are cultured in a medium comprising a carbohydrate source;
• optionally isolating the 3-hydroxyisobutyric acid from the nutrient medium;
• optionally neutralizing the 3-hydroxyisobutyric acid;
• and
• dehydrating the 3-hydroxyisobutyric acid, to obtain methacrylic acid and optionally,
• esterifying the methacrylic acid;
• wherein the genetically modified cell is Corynebacterium glutamicum,
• wherein the enzyme E1 is a methylmalonyl-coenzyme A mutase of EC 5.4.99.2,
• the enzyme E2 is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17) which catalyzes the conversion of methylmalonyl-coenzyme A into methyl malonate;
• the enzyme E3 is an aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase (EC 1.2.3.1);
• and
• the enzyme E4 is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31).