<jats:title>Summary</jats:title><jats:p>Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab‐causing <jats:italic>Streptomyces</jats:italic> species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non‐ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4‐nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4‐nitrotryptophan represents a novel substrate for non‐ribosomal peptide synthetases. Through identification of <jats:italic>N</jats:italic>‐methyl‐4‐nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non‐ribosomal peptide synthetase module specific for 4‐nitrotryptophan.