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Most native producers of ribosomally synthesized and post-translationally modified peptides (RiPPs) utilize N-terminal leader peptides to avoid potential cytotoxicity of mature products to the hosts. Unfortunately, the native machinery of leader peptide removal is often difficult to reconstitute in heterologous hosts. Here we devised a general method to produce bioactive lanthipeptides, a major class of RiPP molecules, in Escherichia coli colonies using synthetic biology principles, where leader peptide removal is programmed temporally by protease compartmentalization and inducible cell autolysis. We demonstrated the method for producing two lantibiotics, haloduracin and lacticin 481, and performed analog screening for haloduracin. This method enables facile, high throughput discovery, characterization, and engineering of RiPPs.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs. Ribosomally synthesized and post-translationally modified peptides are a source of antimicrobials. Here, the authors report a platform for the rapid evaluation and characterization of biosynthetic gene clusters that enables the identification of 30 structurally diverse modified peptides, including three showing antimicrobial activities. [ABSTRACT FROM AUTHOR]