<jats:title>Abstract</jats:title><jats:p>An <jats:italic>E. coli</jats:italic> strain with deletions in five transaminases (Δ<jats:italic>aspC</jats:italic> Δ<jats:italic>ilvE</jats:italic> Δ<jats:italic>tyrB</jats:italic> Δ<jats:italic>avtA</jats:italic> Δ<jats:italic>ybfQ</jats:italic>) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide “xenotetrapeptide” from <jats:italic>Xenorhabdus nematophila</jats:italic>, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of <jats:sc>D</jats:sc>‐amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D<jats:sub>2</jats:sub>O.