Precultures of E. coli BL21(DE3) pJZ_Ptrp_pylT_MmPylS (Y384F) PT7 nisA(amber) pRSFDuet-1 nisBC were grown at 37°C in LB medium supplemented with antibiotics and 1% w/v glucose. Target gene expression was conducted at 27.5°C overnight in autoinducing ZYP-5052 medium (Studier, 2005) supplemented with antibiotics. Next, bacterial cells were harvested by centrifugation. Purification of nisin variants was conducted as described earlier using an N-terminally His-tagged peptide leader and Ni-NTA affinity columns (Baumann et al., 2017).