The stalling development of new antibiotics in combination with an increasing human population density and rising antimicrobial resistance (AMR) in bacteria leads to an emerging antibiotic crisis across the globe. Recent studies estimate that 1.27 million deaths in 2019 were attributed to bacterial AMR alone, while multidrug-resistant bacterial infections contributed to 5 million deaths in already diseased patients. Moreover, during the current coronavirus (COVID-19) pandemic, the viral infections were often accompanied by simultaneous infection with bacterial pathogens. Particularly, co-infections with carbapenemaseproducing pathogens increase lethality rates in patients and contributed to the death of over 6 million COVID-19 patients since the pandemic outbreak in 2020. The challenging research towards discovery and development of new antibiotic classes, especially against Gram-negative pathogens causing lifethreatening infections, rarely results in promising candidates. Consequently, the demand for new drugs against pathogenic Gram-negative bacteria, such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa continuously rises. Presently, there are some candidates in pre-clinical and clinical development stages for the treatment of infections caused by Gram-negative pathogens. However, these mostly belong to already known compound classes or they are novel chemical entities attacking common targets, such as the broad-spectrum ribosome inhibitor odilorhabdin, originally produced by Xenorhabdus nematophila. The darobactins, which are also produced by an entomopathogenic bacterium, Photorhabdus khanii, however, are some of the most promising new antibacterials in the last 50 years given they address a novel broad-spectrum target in Gram-negative pathogens. This mechanism of action overcomes common pathogenic resistance and are effective, not only in vitro, but also in vivo. This family of bicyclic heptapeptides belongs to the biosynthetic class of ribosomally synthesised and post-translationally modified peptides (RiPPs). Darobactins distinguish themselves from other new antibiotic candidates, such as the recently published macolacin, as they do not affect human gut commensals of the family Bacteriodes, a beneficial characteristic considering the intended use in humans. Due to the selective binding to the β-barrel of the outer membrane protein (OMP) BamA, darobactin interferes with the integration of natural OMPs into the membrane by mimicking the OMP signalling peptide. As a consequence, BamA is unable to close the lateral gate and, thus, affects mechanical, kinetic, and energetic properties of the entire complex. Highlighting the value of BamA as a worthwhile antibacterial target, darobactin-resistant E. coli mutants without BamA generated in vitro resulted in substantially reduced fitness and virulence. This indicates a lack of pre-existing resistance and a reduced risk of fast resistance development, at least in E. coli. Darobactin has only been produced in low amounts in the native host bacterium, insufficient for larger industrial fermentation. Total synthesis via multiple steps with relatively low yields was only very recently developed for darobactin A (DA) and requires the usage of toxic chemicals. Excitingly, recently published heterologous expression platforms of the darobactin biosynthetic gene cluster (BGC) offer reasonable production rates in E. coli strains in low cost standard cultivation media. The darobactin biosynthetic gene cluster (BGC) consists of two genes essential for production. One encodes the precursor peptide DarA and the other encodes the radical S-adenosylmethionine (rSAM) enzyme DarE, catalysing the bicyclic crosslinking. Heterologous production rates of up to 25 mg/L in the case of DA and 3 mg/L for the more active D9, generated via biosynthetic pathway engineering, open the door for efficient large-scale production. While the cryo-EM structure of DA in complex with BamA already exists, no structure-activity relationship (SAR) rationale has been reported. Herein, we describe a SAR study inspired by the structure elucidation of the BamABCDE (BAM)-D9 complex via cryo-EM, which aided the design of new darobactin core peptide sequences in vitro. A new, fast, and less expensive core peptide modification strategy, based on the overlap-extension polymerase chain reaction (OE-PCR) method facilitated the generation of fifteen new darA gene variants, including multiple halogenated tryptophans achieved through feeding experimentation, leading to promising derivatives with improved bioactivity and altered core sequences compared to DA, while conserving both tryptophan moieties critical for bicyclisation. Most notably, D22 shows significantly improved MIC values against critical pathogens, such as clinical isolates of carbapenem-resistant A. baumannii (up to 32-fold) and P. aeruginosa (up to 4-fold). Single-particle cryo-EM structure elucidation explained the binding behaviour of the most active new derivative compared to established DA and D9.