To elaborate if increased ncAA concentrations can improve the production of nisin(BocK) variants, L. lactis cultures were supplemented with 0, 1, 2, and 5 mM BocK. As expected, the antimicrobial activity of WT nisin was not influenced by different BocK concentrations. Interestingly, for all tested nisin(BocK) variants, 2 mM BocK delivered the highest antimicrobial activity. Again, MALDI-TOF analysis confirmed the correct peptide mass (compare Figure 5). In conclusion, incorporation of ncAAs into nisin with L. lactis as expression host is possible and highly specific. Still, the in vivo synthesized amount of the ncAA-modified RiPP variants needs further improvement. Despite the low peptide yields, we could demonstrate the functional transfer of the PylRS-based SCS machinery to the Gram-positive host L. lactis.