An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) 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 Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D‐amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D2O. No TATATATATA: An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was unable to degrade several amino acids. This strain was used as expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel “xenotetrapeptide” from Xenorhabdus nematophila.