Since SCS represents a method predominantly described for E. coli strains with single (Wang et al., 2001) or multiple ncAA incorporation and with PylRS expression adopted to this expression host (Sugawara and Nikaido, 2014; Baumann et al., 2018), it was at first glance the promising host of choice for realizing ncAA-modified nisin. Additionally, in-depth analysis of PylRS–tRNAPyl expression in E. coli revealed CUA as the ideal tRNAPyl anticodon for ribosomal incorporation of BocK into growing polypeptide chains (Odoi et al., 2013). The production of post-translationally modified active nisin in E. coli was already documented several times (Shi et al., 2011; Baumann et al., 2017). Moreover, the type II lantibiotic haloduracin was expressed with the required PTM enzymes and in parallel, p-benzoyl-L-phenylalanine was incorporated by SCS (Shi et al., 2011). Recently, the first SCS approach with nisin in E. coli was documented (Zambaldo et al., 2017). In contrast to our general approach which aimed to find the optimal ncAA location resulting in the highest antimicrobial activity, the latter focused only on certain selected serine positions. Aiming for the construction of new ring topologies, these serines were replaced by phenylalanine analogs. However, this did not yield bioactive molecules. In our case, the obtained new-to-nature nisin(BocK) variants were bioactive and modified by both the ncAA and the natural PTM machinery.