Since nisABTC originate from L. lactis and most lantibiotics are originally synthetized by Gram-positive bacteria (Dischinger et al., 2014), L. lactis as expression host for PylRS–tRNAPyl represented a logical step. This is supported by previous studies, which demonstrated that L. lactis is a good candidate for orthogonal expression of functional modification enzymes, e.g., GdmD of Staphylococcus gallinarum (van Heel et al., 2013). This illustrates the high combinatorial possibilities of different PTMs in these organisms and their potential for synthesis of new antibiotics (Montalbán-López et al., 2017). With PylRS–tRNAPyl, we expressed for the first time a tRNA synthetase with its cognate tRNA of archaeal origin in L. lactis. The functionality of this orthogonal pair in this host could also be shown in combination with the NisBTC nisin modification enzymes, leading to PTM of the ncAA-modified nisin precursor and transport out of the cell into the culture supernatant. The high substrate tolerance of the NisBTC system allows modification not only of RiPPs from different origins (van Heel et al., 2016), but also of the newly synthesized nisin(BocK) variants. With our results for L. lactis and previous works on B. cereus (Luo et al., 2016) and Streptomyces albus (Lopatniuk et al., 2017), SCS has now been successfully implemented in three Gram-positive organisms from different prokaryotic families. In all three cases, expression of an OTS was successfully combined with the natural PTM enzymes for either nisin (current study), thiocillin (Luo et al., 2016), or cinnamycin (Lopatniuk et al., 2017) of the expression host. These findings indicate the potential of Gram positives as production hosts for new peptide antibiotics. Concerning the location of ncAA incorporation, especially nisinK12 represents an interesting position for mutagenesis. In accordance with our data, previous studies revealed that replacement of K12 by alanine, serine, or threonine improves the antimicrobial activity of nisin against diverse pathogens, e.g., Enterococcus faecalis, B. cereus, and S. aureus (Molloy et al., 2013). The antimicrobial activity depends on the chemistry of the chosen amino acids, since replacement of K12 by aspartate combined with four other replacements of amino acids by negatively charged ones led to a tremendous decrease of the antimicrobial activity (Khusainov and Kuipers, 2013).