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

Abstract

SUMMARY Thiopeptide 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 7 residues of the prepeptide coding region, enabling the generation of 133 potential thiopeptide variants. Variant libraries were subsequently queried in two ways. First, high through-put MALDI-TOF mass spectrometry was applied to colony-level expressions to sample mutants which 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 were found to produce mature compound, 12 of which retained antibiotic activity and one which had improved activity against Methicillin-resistant Staphylococcus aureus (MRSA).

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

“…(14–17) Biosynthetic engineering of thiopeptides has rapidly emerged as an effective strategy to provide analogs that could ultimately support improved pharmacological and pharmacokinetic parameters. (18–24)…”

The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds

“…We have been among the groups that have reported gene clusters for the ribosome-targeting 26-membered macrocycle class (thiocillins) (28) and the EF-Tutargeting 29-membered macrocycles (GE37468) (17,18). In this study, we have turned to the 35-membered macrocycle scaffold and identified a candidate biosynthetic gene cluster for berninamycin from a draft genome sequence of the known producer Streptomyces bernensis UC 5144.…”

Recombinant thiopeptides containing noncanonical amino acids

“…This method may be generalized by using one of several "photo-caged" unnatural amino acids that have been developed to incorporate natural amino acids that are protected from posttranslational modification in similar fashion and subsequently postbiosynthetically deprotected to yield novel thiopeptides not otherwise expressible in vivo. Although no mutants were found with enhanced activity in this study, new chemical functionalities provided by ncAAs to thiopeptide biosynthesis offer the potential to screen a diverse chemical space for mutants with increased activity in the future (8,13).…”

“…Several methods have been used to produce thiopeptide variants, including biosynthetic pathway engineering (7,8), semisynthesis (9), and total chemical synthesis (10), with the latter providing the greatest control over chemical structure. However, the total synthesis of variants in sufficient quantities for mechanistic or therapeutic purposes is often time-consuming and costly.…”