Antibiotic resistance is a growing health issue, and novel peptide antibiotics like non-ribosomal peptides (NRPs) potentially play a key role in countering bacteria that are resistant to existing antibiotics. However, NRPs are not gene-encoded, making it difficult to generate and screen mutant libraries. In contrast, biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) offers a gene-encoded platform enabling diverse modifications that can mimic NRP structures. By engineering peptide sequences and utilizing RiPP-modifying enzymes, it is possible to create tailor-made antimicrobial peptides. This highlights the potential of RiPP-based systems for activity-based screening and the discovery of novel antibiotics. In this study, Escherichia coli surface display and co-culture techniques were employed to extracellularly produce intracellularly modified RiPPs. After intracellular modification by OspR to introduce ornithine at selected positions and SyncM to introduce a methyllanthionine ring (MeLan), a modified precursor Brevicidine-mimicking mutant peptide (Bre-re-mutant) was displayed by the surface display proteins of Lpp-OmpA and Omp1. In separate cells leader peptidase LahT150 was displayed with InaK. InaK is a type of ice nucleating protein, which enables to display relatively large proteins. Following co-culture of E. coli expressing and displaying the Bre-re-mutant with E. coli expressing the displayed leader peptidase LahT150, the aimed for modified peptide was successfully detected in the supernatant and its antimicrobial activity was assessed. Additionally, the display efficiency of Lpp-OmpA and Omp1 was evaluated, showing a maximum display efficiency of over 90%. This method shows potential for the quick and accurate assessment of in E. coli modified peptides and their antimicrobial activity, facilitating advancements in research and eventual industrial applications.
Xu et al. (Sun,) studied this question.