Antimicrobial activity assay and LC–MS analysis of ncAA-modified nisin produced by SCS in E. coli. (A) Antimicrobial activity of recombinantly produced nisin variants against the nisin-sensitive indicator strain L. lactis NZ9000 pNZnisPT pIL253 (this strain expresses NisP, which catalyzes removal of the nisin leader peptide). As indicated, recombinant expression of both nisA(amber) variants was conducted in presence or in absence of BocK. Fifty microliters of E. coli cell lysate normalized by harvested cell density was used. Cm served as antimicrobial control compound. (B) ESI–MS deconvolution chromatogram for nisin(I4BocK) and nisin(K12BocK) samples purified via IMAC. Theoretical [M+Na]+ masses for the ncAA-modified prepeptides (still carrying the leader) after PTM by NisBC are 7428.46 and 7413.45 Da, respectively. Observed masses: 7428.40 and 7413.47 Da. Chromatogram shown for nisin(I4BocK), see Supplementary Figure S3B for that of nisin(K12BocK).