Preceding darobactin SAR studies focused on native darobactins or were based upon observations from them. Elucidation of the MoB of DA and D9 to BamA, combined with structural data of recently published darobactins, enabled a more guided approach, in which we constructed twenty new derivatives, including twelve derivatives with terminal L-tryptophan exhibiting similar or better activity than DA (Figure 3). According to our findings, structural changes at position 4 appears to have only a minor influence, while positions 6 and 7, necessary for interactions with β-hairpins of BamA, seem to have a major influence on activity and could be used as a potential starting point for improvement of pharmaceutical properties through structural changes. The most promising compound developed to date, based on our SAR study is the biosynthetically engineered new darobactin D22, which surpasses the antibacterial activity of all identified native darobactins. This is remarkable, as natural products are produced in co-evolution over millions of years, resulting in structural diversity and target specificity for fungi, eukaryotes and bacteria. The engineered derivative D22 is highly active against a broad range of Gram-negative bacterial pathogens demonstrating superiority even to the previous, biosynthetically engineered frontrunner D9, which already surpassed the activity of originally reported native DA. In particular, D22 displays bactericidal activity against hard-to-treat CRAB and is equipotent to the highly active, yet nephrotoxic, last resort antibiotic colistin. Furthermore, D22 was shown to be non-toxic in initial assays in vitro (HepG2) and in vivo (zebrafish larvae). Further studies will explore the optimised effects of D22 regarding mechanistic, kinetic, and energetic properties, e.g. regarding the linker region and the eight β-hairpins, as detailed by Ritzmann et al. for DA. Future studies should also include a comparison to the structurally variable BAM complex of A. baumannii strains, of which a costructure with darobactins has not yet been solved. However, our activity and toxicity data of D22 are encouraging to advance into further preclinical studies. While currently established protocols for cultivation in E. coli are favourable for fast and robust generation of novel, even halogenated, darobactins in quantities suitable for research and development purposes, they are hard-pressed to satisfy supply demands for such future studies, because of low self-resistance of the Gram-negative heterologous host E. coli. Changing the heterologous producer to a Grampositive strain such as Bacillus subtilis, which has been successfully applied in industrial fermentations, might enable higher darobactin titres. Alternatively, very recently published total syntheses reported for DA may be applicable but currently suffer from yield issues as well. To summarise, our SAR approach presented structural knowledge about the MoB of the darobactins and resulted in novel derivatives with up to 32-fold increased activity against CRAB, compared to the most potent known derivative D9. The new, distinguished D22 was found to be comparable to clinically used antibiotics in its activity against certain Gram-negative pathogens. Consequently, our approach led to a new frontrunner molecule of darobactins showing more favourable antibacterial activity. Compound D22 is particularly active against critically prioritised Gram-negative pathogens encouraging further preclinical studies with this highly promising scaffold to hopefully and eventually achieve proof of efficacy in humans for this exciting and promising molecule class.