In a previous study, recombinant production of fully modified nisin was established in E. coli (Shi et al., 2011). Based on this data, we constructed a T7 promoter-based setup for the recombinant expression of nisABC. The expression of nisA, nisBC, and pylTS from three different plasmids revealed to be a significant metabolic burden for the BL21(DE3) host cells, resulting in slow growth and a low final optical density of the cultures (data not shown). Thus, we reduced the number of required plasmids by introducing nisA(amber) variants into the OTS plasmid yielding pJZ_Ptrp_pylT_MmPylS(Y384F) PT7 nisA(amber). We decided to explore a full amber scanning library, which displays in-frame amber stop codons successively replacing each of the sense codons of the nisin core peptide, to allow for identification of variants with efficient SCS and retaining activity. Following plasmid construction, E. coli was co-transformed by the individual library member plasmids and a compatible second plasmid for NisBC co-expression. The latter two PTM enzymes catalyze the dehydration and circularization of the nisin precursor peptide (Lubelski et al., 2008). Using autoinduction medium, nisABC expression was driven by the T7 promoter system, whereas OTS expression (pylTS) was constitutive.