A scalable platform to discover antimicrobials of ribosomal origin

Abstract

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.

Robotic Construction and Screening of Lanthipeptide Variant Libraries in Escherichia coli

“…Overall, we successfully devised a high-throughput, robotic workflow to create and screen recombinant libraries of RiPP natural products. This workflow may accelerate large-scale (meta)­genome mining to discover new RiPPs, mechanistic investigation informed by massive SAR profiling, and data-driven bioengineering.…”

“…To date, most studies focus on metabolic and genome engineering to improve the biosynthesis of primary metabolism-derived products, but applications on natural products from secondary metabolism are underrepresented. 12,[19][20][21][22]44 The main challenges in automating synthetic biology workflows on natural products are associated with the high-throughput test on molecular structures and bioactivities.…”

“…Biofoundries are a type of automation infrastructure to accelerate and standardize biological design–build–test (DBT) cycles through the integration of robotics, high-throughput instrumentation, and synthetic biology. − Automated workflows have been developed on biofoundry work cells for the rapid design and construction of genetic constructs and engineered microbial strains. To date, most studies focus on metabolic and genome engineering to improve the biosynthesis of primary metabolism-derived products, but applications on natural products from secondary metabolism are underrepresented. ,− , The main challenges in automating synthetic biology workflows on natural products are associated with the high-throughput test on molecular structures and bioactivities.…”

Improved production of class I lanthipeptides in Escherichia coli

“…The recent development of an automated and high throughput pipeline for RiPP discovery enables the identification of the products of BGCs via the heterologous expression of refactored BGCs in E. coli. 30 While the expression of class II lanthipeptide BGCs exhibited the highest success rate of all RiPPs investigated and resulted in the discovery of new compounds active against ESKAPE pathogens, the expression of class I lanthipeptide BGCs encountered two major challenges that hampered further structural and biological studies. 30 Although heterologous expression of a number of class I lanthipeptides has been successful in E. coli, [31][32][33][34][35][36][37] many class I dehydratases fail to fully modify their substrates in this heterologous host.…”

EngineeringEscherichia colito utilize erythritol as sole carbon source

“…In metabolic engineering and synthetic biology, Escherichia coli has been engineered for a broad range of applications with a main focus on the production of valuable products [26][27][28] .…”