Towards Biocontained Cell Factories: An Evolutionarily Adapted Escherichia coliStrain Produces a New-to-nature Bioactive Lantibiotic ContainingThienopyrrole-Alanine

Genetic code engineering that enables reassignment of genetic codons to non-canonicalamino acids (ncAAs) is a powerful strategy for enhancing ribosomally synthesizedpeptides and proteins with functions not commonly found in Nature. Here we reportthe expression of a ribosomally synthesized and post-translationally modifiedpeptide (RiPP), the 32-mer lantibiotic lichenicidin with a canonical tryptophan(Trp) residue replaced by the ncAA L- β-(thieno[3,2- b ]pyrrolyl)alanine ([3,2]Tpa) which doesnot sustain cell growth in the culture. We have demonstrated that cellular toxicityof [3,2]Tpa for the production of the new-to-nature bioactive congener oflichenicidin in the host Escherichia coli can be alleviated by using anevolutionarily adapted host strain MT21 which not only tolerates [3,2]Tpa but alsouses it as a proteome-wide synthetic building block. This work underscores thefeasibility of the biocontainment concept and establishes a general framework fordesign and large scale production of RiPPs with evolutionarily adapted hoststrains.