Fermentative -Methylanthranilate Production by Engineered .

The -functionalized amino acid -methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid -isopropyl -methylanthranilate, the flavor compound -methyl--methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to -alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium are operated industrially at the million tons per annum scale. Fermentative processes using for -alkylated amino acids based on an imine reductase have been developed, while -alkylation of the aromatic amino acid anthranilate with -adenosyl methionine as methyl-donor has not been described for this bacterium. After metabolic engineering for enhanced supply of anthranilate by channeling carbon flux into the shikimate pathway, preventing by-product formation and enhancing sugar uptake, heterologous expression of the gene encoding anthranilate -methyltransferase from resulted in production of -methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene improved -methylanthranilate production. In a test bioreactor culture, the metabolically engineered C1* strain produced NMA to a final titer of 0.5 g·L with a volumetric productivity of 0.01 g·L·h and a yield of 4.8 mg·g glucose.