Following established procedures, efforts were made to quantify and optimize ncAA-containing nisin variant production. Via Coomassie-stained PAGE, it was observed that peptide quantities and purities were lower than reported earlier (Shi et al., 2011) (data not shown). Yields of recombinant production were reduced upon ncAA incorporation as commonly is the case for SCS (Zheng et al., 2016). It should be noted that recombinant production and SCS were performed in a release factor-1 (RF-1) positive E. coli B laboratory strain [BL21(DE3)], where amber SCS competes with translation termination. Nevertheless, the PylRS-based system can deliver efficient amber suppression in this strain as long as a single in-frame stop codon is used (Odoi et al., 2013). Furthermore, E. coli BL21(DE3) was chosen as it grows robustly to high cell density in the nisin biosynthesis setup (data not shown). Despite the genetic complexity of the system, the feasibility of recombinant ncAA-containing nisin production was demonstrated. Employing the WT L. lactis PTM enzymes for prepeptide processing, positions were identified to allow for BocK incorporation into nisin – leading to novel bioactive ncAA-modified RiPPs. Since nisin(I4BocK) and nisin(K12BocK) represented reasonable antimicrobial activity, they were chosen as candidates to transfer this OTS into the natural production host of nisin, L. lactis.