Robotic Construction and Screening of Lanthipeptide Variant Libraries in Escherichia coli

Lanthipeptides are a major class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by thioether cross-links called lanthionine (Lan) and methyllanthionine (MeLan). Previously, we developed a method to produce mature lanthipeptides in recombinant Escherichia coli, but manual steps hinder large-scale analogue screening. Here we devised an automated workflow for creating and screening variant libraries of haloduracin, a two-component class II lanthipeptide. An integrated work cell of a synthetic biology foundry was programmed to robotically execute DNA library construction, host transformation, peptide production, mass spectrometry analysis, and activity screening by agar diffusion assay. For recombinantly produced Hal alpha peptides, the sequence-activity relationship of 380 single-residue variants and >1300 triple-residue combinatorial variants were rapidly analyzed in microplates within weeks. The peptide expression levels in E. coli were also visualized via robotic creation and analysis of GFP-lanthipeptide fusions for select peptide mutants. Following shake-flask fermentation and purification, one Hal alpha mutant was confirmed with enhanced specific antimicrobial activity relative to the wild-type peptide. Overall, this approach may be generally applicable for the high-throughput characterization and engineering of RiPP natural products.