FermentativeN-Methylanthranilate Production by EngineeredCorynebacterium glutamicum

TheN-functionalized amino acidN-methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloidO-isopropylN-methylanthranilate, the flavor compoundO-methyl-N-methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes toN-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 bacteriumCorynebacterium glutamicumare operated industrially at the million tons per annum scale. Fermentative processes usingC. glutamicumforN-alkylated amino acids based on an imine reductase have been developed, whileN-alkylation of the aromatic amino acid anthranilate withS-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 geneanmtencoding anthranilateN-methyltransferase fromRuta graveolensresulted in production ofN-methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase genesahHimprovedN-methylanthranilate production. In a test bioreactor culture, the metabolically engineeredC. glutamicumC1* strain produced NMA to a final titer of 0.5 g center dot L(-1)with a volumetric productivity of 0.01 g center dot L-1 center dot h(-1)and a yield of 4.8 mg center dot g(-1)glucose.