After establishing a total synthesis route to avoid dependance on the low yield fermentation process and elucidation of the absolute configuration of Corramycin, we planned to further investigate the potential of Corramycin as a starting point for the development of a new antibacterial drug. Therefore, we first performed a time-kill curve experiment at 4x and 8x MIC of Corramycin using E. coli ATCC25922 cultivated in the MHB rich medium or in the M9 minimal medium, (Supplementary Figure S 68). This experiment revealed that Corramycin is rapidly bactericidal, independent of the medium used. However, we observed re-growth in MHB after 6 hours, indicating rapid selection of mutants resistant to Corramycin, with a high-frequency of resistance (FoR) under these conditions. Therefore, we performed a FoR experiment using E. coli ATCC25922 in MHB and M9 medium at 4x MIC of Corramycin. Importantly, the FoR was much higher in MHB medium (3.8 × 10 -6 ) compared to M9 medium (3 × 10 -10 ) (Supplementary Table ). Next, we sequenced the genome of the resistant E. coli clones cultivated in MHB medium to identify potentially responsible mutations. A frameshift in the gene encoding the SbmA transporter gave a first hint about the bacterial uptake systems of Corramycin since the potential inactivation of SbmA, caused by the frameshift, may confer resistance towards Corramycin. Interestingly, clones harboring the mutated SbmA were only resistant towards Corramycin in MHB medium but not in M9 medium. Thus, we performed an additional resistance experiment with SbmA mutants in M9 medium. We sequenced the genome of clones which gained resistance towards Corramycin in M9 medium and found additional mutations (frameshifts) in YejE or YejF. Independent or combined deletions of SbmA and YejEF in E. coli ATCC25922 generated using λ red-mediated homologous recombination (see SI) resulted in strains that showed cultivation medium-dependent resistance towards Corramycin (Table ). The uptake appears to be dependent solely on the SbmA transporter in MHB medium and on both SbmA and YejEF transporters in M9 medium. Functional complementation of SbmA and YejEF in E. coli ATCC25922 ∆sbmA/∆yejEF revealed that solely SbmA restores the susceptibility to Corramycin in MHB medium. Notably, in case of YejEF complementation, we introduced the entire operon yejABEF to ensure a balanced coexpression of all genes regulated by the same promoter system. One recognized bottleneck for the identification of novel agents with specific antimicrobial activity against Gram-negative bacteria is the difficulty for compounds to penetrate the robust permeability barrier caused by the differing properties of the outer and inner membranes. Corramycin is able to hijack several bacterial transporters in a seemingly redundant manner. SbmA is an ABC transporter that has been described as responsible for the uptake of several antibacterial molecules such as Microcin J25 and Bleomycin. SbmA is present in E. coli and K. pneumoniae but absent in other Enterobacteriaceae where an ortholog can be identified under the name of YddA. YejABEF is an inner membrane ABC transporter, which is conserved in Gram-negative bacteria and has been shown to be responsible for the uptake of microcin C. Here, both transporters, SbmA and YejABDEF, have been shown to play a vital role in the uptake of Corramycin. However, SbmA seemed to be the main uptake system when E. coli was cultivated in MHB medium. The dominant role of SbmA for Corramycin susceptibility in MHB medium was also confirmed using other E. coli strains, such as K12, ATCC35218 and BAA2469 (data not shown). Since the transport and accumulation of compounds in bacteria is one of the major bottlenecks in antibiotic development, potential drugs hijacking only a single transporter make quick resistance development likely, which was, e.g., shown for Fosfomycin, Kazugamycin and GE81112. This was also shown here, when SbmA is the only transporter leading to Corramycin uptake in MHB medium. The high FoR against Corramycin observed in MHB medium could have been potentially an exclusion criterion regarding the further development of Corramycin towards a drug discovery program. However, the FoR was much lower in M9 minimal medium, where two transporter systems had to be mutated to decrease Corramycin susceptibility. As bacterial resistance development in minimal media is thought to be more mimetic to an actual infection in vivo, we decided to test the in vivo efficacy of Corramycin in infected mice, despite the poor FoR experiment results in the rich medium. The mice were first infected intraperitoneally with E. coli ATCC35218 and subsequently treated with different doses of Corramycin ranging from a total of 10 to 30 mg per kg body weight divided into two doses, 1 h and 3 h after infection. We observed a remarkable dose-dependent reduction of the bacterial load 4 h after infection resulting in survival of the mice up to 60 % after 4 days starting from the 20 mg kg -1 total dose (Figure ). In comparison, 0 % of survival was observed for the control mice, which were treated with the vehicle substance. Of note, Corramycin is rapidly eliminated after i.v. administration, keeping the plasma concentration above the MIC for less than 30 min. However, despite this rapid elimination, the antibacterial effect was strong enough in the animals to achieve survival after a single day of treatment.