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

Lanthipeptides are a major group of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by microorganisms, have extensive therapeutic and industrial applications, therefore, discovery of novel lanthipeptides and increasing production are the research hotspots in the synthetic biology and metabolic engineering fields. 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.

In this work, we developed a cell-free protein synthesis (CFPS) platform and demonstrated its use for discovering novel lanthipeptides and guiding lanthipeptides overproduction in vivo using nisin as an example. For discovery of novel lanthipeptides, we mined all nisin analogs (~210) from NCBI database, then deduplication and excluded known lanthipeptides and found 18 potential lanthipeptides, they were mined in our CFPS platform in a single day and identified 4 novel antibacterial lanthipeptides (one of them show better antibacterial activity than nisin). Next, we used antimicrobial activity screening in CFPS platform for discovering nisin mutants against the growth of gram-negative bacterial, a nisin mutant library with anti-bacterial activity was screened and achieved one mutant from 3,000 mutants with better anti-E. coli activity than nisin.

For overproduction of lanthipeptides, we identified NisB (the dehydrase in nisin biosynthesis) is the key system controller in nisin biosynthesis in the CFPS platform. Then the industrial nisin Z producing strain was precisely improved by target metabolic engineering in vivo (precursor peptide gene of nisin and nisB overexpression), the titer of engineered strain is 8,828 IU/ml (60% nisin Z increase than the current industrial nisin strain. This strategy (nisB overexpression) was expanded to enhance nisin analogs production in E. coli, an 89.2% increase in nisin analogs was achieved. The newly developed CFPS platform has the potential to substantially expand RiPPs research and production.