The glycopeptide antibiotics (GPAs, including vancomycin and teicoplanin) are highly cross-linked heptapeptide antibiotics that are assembled in a modular fashion through non-ribosomal peptide synthesis. The production of a linear peptide precursor is followed by a cyclisation cascade of three or four cytochrome P450 enzymes that occurs late in biosynthesis, with the exact timing and peptide structure when cross-linking occurs previously unclear. Recently, Max Cryle and his group at Monash University, working with German collaborators, have shown that differences in results from in vitro and in vivo studies on these systems can be explained by the surprising tolerance of the final peptide-bond-forming domain for altered peptide substrates (Schoppet M., Peschke M., Kirchberg A., Weibach V., Süssmuth R.D., Stegmann E., Cryle M.J. Chem. Sci. 2019, 10, 118–33). The substrate tolerance displayed by this domain during peptide biosynthesis has important implications for the ability to undertake biosynthetic redesign of such systems via the incorporation of different amino acid monomers. Furthermore, it explains why the alteration of non-ribosomal peptide synthetase assembly lines in vivo can lead to the accumulation of off-pathway intermediates that are not normally seen during wildtype biosynthesis.