To determine if the same ncAA can also be incorporated into nisin, gfp(amber) was replaced by nisA(amber). As target locations in nisin, the codons for core peptide I4 and K12 were chosen, as they were promising candidates as indicated by the E. coli experiments (see above). To synthesize the variants and to purify them from the culture supernatant, L. lactis NZ9000 transformed with pIL3EryBTC and pNZ-RBSpylTSnisA(I4amber) or pNZ-RBSpylTSnisA(K12amber), respectively, was cultivated. These strains express the transporter NisT capable to transport nisin out of the cells, which enables peptide purification from the culture supernatant. The nisin variants remain inactive at this point, because no protease is present to cleave off the leader peptide. The antimicrobial activity of the supernatant of the corresponding cultures was checked against the nisin-sensitive indicator strain L. lactis NZ9000 pNZnisPT pIL253. By using this NisP producing indicator strain, the leader peptide gets cleaved off, liberating active nisin (Khusainov et al., 2011). As seen before for samples purified from E. coli, only samples from production cultures supplemented with BocK showed antimicrobial activity, indicating that translation was terminated in absence of ncAA supplementation and that no canonical amino acid was incorporated instead. Three internal controls were utilized: cells bearing pNZ-RBSpylTSnisA pIL3EryBTC served as positive control to determine the influence of the rearranged promoter on nisin WT production. Culture supernatant of these cells always showed antimicrobial activity independent of BocK addition, assuring the general functionality of the nisin production system. Two internal negative controls were performed. Supernatant of cells expressing only PylRS–tRNAPyl either in the presence and absence of BocK never had any antimicrobial effect. Consequently, antimicrobial activity was caused by the novel nisin variants and not by the orthogonal tRNA synthetase–tRNA pair or the supplemented ncAA (Figure 4).