A cell-free platform based on nisin biosynthesis for discovering novel lanthipeptides and guiding their overproductionin vivo

Abstract

Lanthipeptides have extensive therapeutic and industrial applications; however, since many are bactericidal, traditional in vivo platforms are limited in their capacity to discover and mass produce novel lanthipeptides as bacterial organisms are often critical components in these systems. We developed a cell-free protein synthesis (CFPS) platform that enables rapid genome mining, screening and guiding overproduction of lanthipeptides in vivo. For proofof-concept studies, the type I lanthipeptide, nisin, was selected. Four novel lanthipeptides with anti-bacterial activity were identified among all nisin analogs in the NCBI database in a single day. Further, we coupled the CFPS platform with a screening assay for gram-negative bacterial growth and identified a potent nisin mutant, M5. The titer of nisin and nisin analogs significantly improved with CFPS platform guidance. Owing to the similarities in biosynthesis, our CFPS platform is broadly applicable to other lanthipeptides, provides a universal method for lanthipeptides discovery and overproduction.

Cell‐Free Exploration of the Natural Product Chemical Space

“…Lastly, the pathway for the prototypical lanthipeptide, nisin, was recently rebuilt using extract-based CFE. [50] The authors coupled cell-free nisin biosynthesis with an antibiotic activity assay which allowed them to screen over 3000 analogues and quickly identify two variants that were more active than the parent compound. Cell-free systems have been used to synthesize other ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, however only precursor peptides (or analogues) were synthesized by CFE.…”

Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing

“…Cell-free systems are derived from the soluble components of crude cell lysates (often referred to as cell extracts) that contain active biological machinery for metabolism, transcription, and translation without homeostasis or growth constraints faced by living cells. These systems have emerged as powerful tools for studying and harnessing protein and metabolite synthesis. − Applications span prototyping genetic parts or metabolic pathways, − biomanufacturing small molecules, − synthesis of proteins, , molecular diagnostics, − and educational kits. − …”