Following recombinant expression of pylTS and nisABC in E. coli, conducted both in presence and in absence of BocK (Figure 1B), the cell lysates were examined for antimicrobial activity. For this, the nisin-sensitive NisPT-expressing L. lactis strain NZ9000 pNZnisPT pIL253 (Khusainov et al., 2011) was used, which is an indicator strain capable to cleave the leader peptide to yield fully mature active nisin. Although mature WT nisin is ineffective against the Gram-negative bacterium E. coli (Helander and Mattila-Sandholm, 2000), a nisA leader carrying an N-terminal His-tag was utilized in order to facilitate purification of the produced peptides. This modification does not influence the removal of the leader peptide, since the WT NisP cleavage sequence ASPR | IT remains intact (Plat et al., 2011; Lagedroste et al., 2017; Montalban-Lopez et al., 2018). Screening of the amber-scanned nisA library was performed. Targeting residues corresponding to the first two rings in the mature lantibiotic (i.e., I1 until K12, compare Figure 1A) revealed promising candidates, where ncAA-dependent antimicrobial activity was observed. Distinct inhibition of microbial growth was observed for samples corresponding to nisin(I4BocK) and nisin(K12BocK), whereas very low or no inhibition, respectively, took place in absence of ncAA supplementation (Figure 2A). Noteworthy, SCS at most positions within the prepeptide did not result in bioactivity (Supplementary Figure S2). With no or little change upon ncAA (BocK) addition, activity was also observed when the amber stop codon was placed close to the 3′-end of nisA, as shown for the penultimate prepeptide position in construct nisA(S33amber) (Supplementary Figure S2). The latter is in line with observations made from production of modified/truncated variants in L. lactis: also those truncated prepeptides become processed by NisBC in vivo and attain detectable levels of antimicrobial activity (Rink et al., 2007).