To express tRNAPyl and PylRS in L. lactis, pylTS were amplified from plasmid pJZ_Ptrp_pylT_strep-MmPylS(Y384F) with the primers PylTSfwNco and PylTSrevKpn. The PCR product and the vector pLGΔRBS-GFP were digested with the restriction enzymes NcoI and KpnI and ligated, resulting in vector pNZ-RBSpylTS. Subsequently, selected nisA(amber) variants were amplified by the primer pair NisLibraryfwKpn and NisLibraryrevXba. The resulting PCR products and the vector pNZ-RBSpylTS were digested by KpnI and XbaI, and the fragments were assembled to form plasmid pNZ-RBSpylTSnisA(amber). To synthesize the nisA WT control, the amber codon of pET-21a_P_His6_leader_nisA(K34amber) was replaced by the WT lysine codon using the mutagenesis primer NisAWTrevXba, while as forward primer NisLibraryfwKpn was utilized. The further cloning procedure was identical and resulted in the plasmid pNZ-RBSpylTSnisA. To enable the PTMs of the nisin precursor (dehydration and cyclization followed by transport out of the cell), L. lactis NZ9000 pIL3EryBTC encoding the modification enzymes NisBTC was transformed by the nisA plasmid constructs.