| Summary: | <i>N-</i>alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for <i>N-</i>alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to <i>N-</i>alkylated amino acids like <i>N-</i>methyl-<span style="font-variant: small-caps;">l</span>-alanine or <i>N-</i>methylantranilate, a precursor of acridone alkaloids, have been established using engineered <i>Corynebacterium glutamicum</i>, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of <i>C. glutamicum</i> for de novo production of <i>N-</i>methylphenylalanine based on reductive methylamination of phenylpyruvate. <i>Pseudomonas putida</i> Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes <i>trpEG</i> and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of <span style="font-variant: small-caps;">l</span>-tryptophan and <span style="font-variant: small-caps;">l</span>-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of <i>DpkA</i><sup>P262A,M141L</sup>, <i>N-</i>methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an <i>N-</i>methylphenylalanine titer of 0.73 ± 0.05 g L<sup>−1</sup>, a volumetric productivity of 0.01 g L<sup>−1</sup> h<sup>−1</sup> and a yield of 0.052 g g<sup>−1</sup> glucose were reached. When xylose isomerase gene <i>xylA</i> from <i>Xanthomonas campestris</i> and the endogenous xylulokinase gene <i>xylB</i> were expressed in addition, xylose as sole carbon source supported production of <i>N-</i>methylphenylalanine to a titer of 0.6 ± 0.04 g L<sup>−1</sup> with a volumetric productivity of 0.008 g L<sup>−1</sup> h<sup>−1</sup> and a yield of 0.05 g g<sup>−1</sup> xylose. Thus, a fermentative route to sustainable production of <i>N-</i>methylphenylalanine by recombinant <i>C. glutamicum</i> has been established.
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