Fig. 4. Synthesis of lactazole-like thiopeptides with randomized sequences.

a LazA^min variants containing ten consecutively randomized amino acids were first treated with LazDEF and then with LazBC, tRNA^Glu, and GluRS (see Methods for details), and the outcomes were analyzed by LC-MS. Displayed are LC-MS chromatograms (^brEIC on the left showing partially processed linear peptides and LP-NH₂ after enzymatic treatment, and ^nrEIC chromatograms on the right for expected thiopeptides generated at m/z 0.10 tolerance window). Mutants highlighted in light blue indicate efficient thiopeptide assembly; in yellow, inefficient thiopeptide formation accompanied by the accumulation of linear intermediates and side products. One construct, 10aa-sub4, resulted in eight different thiopeptides, partial annotation of which can be found in Supplementary Fig. 20. Efficient in-vitro biosynthesis observed in nine out of ten cases underscores the substrate plasticity of FIT-Laz. b LC-MS chromatograms as in a for the enzymatic processing of a 34 amino acid-long LazA^min variant on the left with a zoomed-in mass spectrum of the produced thiopeptide on the right.