A Cell‐Free Platform Based on Nisin Biosynthesis for Discovering Novel Lanthipeptides and Guiding their Overproduction In Vivo

Abstract

Lanthipeptides have extensive therapeutic and industrial applications. However, because 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. Herein, the development of a cell‐free protein synthesis (CFPS) platform that enables rapid genome mining, screening, and guided overproduction of lanthipeptides in vivo is described. For proof‐of‐concept studies, a type I lanthipeptide, nisin, is selected. Four novel lanthipeptides with antibacterial activity are identified among all nisin analogs in the National Center for Biotechnology Information (NCBI) database in a single day. Further, the CFPS platform is coupled with a screening assay for anti‐gram‐negative bacteria growth, resulting in the identification of a potent nisin mutant, M5. The titers of nisin and the nisin analog are found to be improved with CFPS platform guidance. Owing to the similarities in biosynthesis, the CFPS platform is broadly applicable to other lanthipeptides, thereby providing a universal method for lanthipeptide discovery and overproduction.

Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance

“…Some examples include 2,3-butanediol, 45 diverse glycan motifs, 46 and a cyclic dipeptide produced from two non-ribosomal peptide synthetases. 47 Several mature RiPPs have been produced using cell-free methods, such as goadsporin, 48 nisin, 49 thiocillin, 50 lactazole 51 . To the best of our knowledge, lasso peptides have not previously been produced in the manner.…”

Biocatalytic synthesis of peptidic natural products and related analogues

“…Furthermore, cell-free protein synthesis (CFPS) platforms have been used to produce peptide substrates for the synthesis of RiPPs ( Figure 7 A). In a recent example, the Liu group developed and optimized the E. coli CFPS system to synthesize the precursor peptide (NisA) and three biosynthetic enzymes (NisB, NisC, and NisP) of one class I lanthipeptide nisin and achieved a yield of 200 IU/mL ( Liu et al., 2020 ). This system also produced four novel lanthipeptides with antibacterial activity from the mined biosynthetic gene clusters within 24 h, clearly stating the potential of this system for quick access to RiPPs and their analogues.…”

Mining and Biosynthesis of Bioactive Lanthipeptides From Microorganisms

“…Nisin, as the first food additive, has a strong inhibitory effect on many G + bacteria including Streptococcus pneumoniae , and also has certain inhibitory activity on some G – bacteria ( Ahmadi et al, 2017 ; Li et al, 2018 ). However, compared to the effects against G + bacteria, most of lanthipeptides show relatively weak inhibitory activity against G – bacteria ( Liu et al, 2020 ).…”

“…Currently, some in vitro cell free biosynthesis platforms were well-designed for mining of lanthipeptides with the superiority of independent of cell growth, especially combined with high-throughput bioactivity screening systems. For instance, by use of CFPS (cell-free protein synthesis) set up based on E. coli cell extract, searching all nisin-related gene sequences in NCBI database resulted in biosynthesis of four new lanthipeptides with antibacterial activity in a single day ( Zhang et al, 2018 ; Liu et al, 2020 ).…”