In this study, we established genetic code expansion of L. lactis by functional expression of an OTS based on PylRS–tRNAPyl. The successful combination of SCS and PTM enzymes led to the production of bioactive BocK-modified nisin variants. To identify the ideal host for genetic code expansion and post-translationally modified lantibiotic production, we tested two expression hosts: E. coli and L. lactis, because each of them has distinct advantages.