| Summary: | The <i>N</i>-functionalized amino acid <i>N</i>-methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid <i>O</i>-isopropyl <i>N</i>-methylanthranilate, the flavor compound <i>O</i>-methyl-<i>N</i>-methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to <i>N</i>-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 <i>Corynebacterium glutamicum</i> are operated industrially at the million tons per annum scale. Fermentative processes using <i>C. glutamicum</i> for <i>N</i>-alkylated amino acids based on an imine reductase have been developed, while <i>N</i>-alkylation of the aromatic amino acid anthranilate with <i>S</i>-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 <i><span style="font-variant: small-caps;">anmt</span></i> encoding anthranilate <i>N</i>-methyltransferase from <i>Ruta graveolens</i> resulted in production of <i>N</i>-methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene <i>sahH</i> improved <i>N</i>-methylanthranilate production. In a test bioreactor culture, the metabolically engineered <i>C. glutamicum</i> C1* strain produced NMA to a final titer of 0.5 g·L<sup>−1</sup> with a volumetric productivity of 0.01 g·L<sup>−1</sup>·h<sup>−1</sup> and a yield of 4.8 mg·g<sup>−1</sup> glucose.
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