Notably, change of the amino acid in position 5 from L-lysine to L-arginine resulted in lower production yields and reduced activity, as shown in our previous publication's extract activity and by analysing pure DD (WNWTRSF) by Böhringer et al. However, this data is contradictory to the darobactin binding mode determined in our cryo-EM experiments. The backbone of L-lysine at position 5 is only involved in interaction with the BamA β1-strand on F426 with its carbonyl-and on A427 by the nitrogen of the amide group (Figure ). The L-lysine sidechain with its terminal amino group is not involved in darobactin-BamA interaction, but is oriented into the exterior region of the β-barrel (Figure Kaur et al. found that the positive charge of lysine sidechain interacts with phosphate moieties of cardiolipin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, which should also hold true for arginine. Accordingly, we were interested in the design of D39 with L-threonine at position 4, L-arginine at position 5 and 6 and L-tryptophan at position 7 (WNWTRRW). The designed darobactin core sequences encoding for D22 to D32 and D36 to D39 were generated by introducing point mutations via OE-PCR in pNOSO-darABCDE-9 (DSM 33802). A detailed explanation can be found in the methods (Figure S 4; Table S 2). Subsequent mass spectrometry analysis (Table ) and bioactivity ranking of the crude extracts for prioritisation of the most active derivatives were performed as described in our previous publication, which led to the discovery of D9. Different molecular ions observable in electrospray ionisation (ESI) mass spectrometry (MS) were used in combined extracted ion chromatograms (EIC) to obtain comparable data for the area under the curve (AUC), since introduction of basic amino acids shifted the most abundant ion species from the doubly charged to the triply charged species (Table ).