In this study, a double tracked approach toward ncAA-modified nisin was followed: First, E. coli was equipped with both the SCS and nisin biosynthesis machineries for recombinant production of nisin modified with Nε-Boc-L-lysine (BocK) as chosen ncAA (Figure 1B). Second, the genetic code of L. lactis, the Gram-positive native nisin production host, was expanded by introducing the PylRS–tRNAPyl pair to enable SCS. Following site-specific incorporation of BocK into different locations of nisin in vivo, the effects on the antimicrobial activity of nisin were analyzed.