Codon Randomization for Rapid Exploration of Chemical Space in Thiopeptide Antibiotic Variants

SummaryThiopeptide antibiotics exhibit a profound level of chemical diversity that is installed through cascades of posttranslational modifications on ribosomal peptides. Here, we present a technique to rapidly explore the chemical space of the thiopeptide GE37468 through codon randomization, yielding insights into thiopeptide maturation as well as structure and activity relationships. In this incarnation of the methodology, we randomized seven residues of the prepeptide-coding region, enabling the generation of 133 potential thiopeptide variants. Variant libraries were subsequently queried in two ways. First, high-throughput MALDI-TOF mass spectrometry was applied to colony-level expressions to sample mutants that permitted full maturation of the antibiotic. Second, the activity of producing mutants was detected in an antibiotic overlay assay. In total, 29 of the 133 variants produced mature compound, 12 of which retained antibiotic activity and 1 that had improved activity.

Graphical AbstractFigure optionsDownload high-quality image (260 K)Download as PowerPoint slideHighlights
► Thiopeptide GE37468 expression was increased 30-fold above previous reports
► Codon randomization enabled rapid exploration of chemical space
► Thirteen thiopeptide analogs had activity against MRSA
► Identification of linear precursor peptides allowed insights into biosynthetic timing