For expression in E. coli, selected variants from the nisA(amber) library (flanked by a T7 promoter and terminator) were PCR-amplified with the primers PT7-NisA_f and NisA T7term_r. Purified PCR products were cloned using SpeI and PstI into pJZ_Ptrp_pylT_strep-MmPylS(Y384F) treated with XbaI and PstI to yield pJZ_Ptrp_pylT_MmPylS(Y384F) PT7 nisA(amber). In order to combine PTM and SCS for the production of ncAA-modified nisin variants, BL21(DE3) cells were co-transformed with the latter plasmid and pRSFDuet-1 nisBC.