To verify the promising activity data of crude extracts, certain derivatives with increased activity were selected for large-scale production and isolation. During the production optimisation of D22, the commercially available E. coli BL21-Gold (DE3), engineered for reduced leaky expression (Agilent Technologies, California, USA), was evaluated and found to be favourable for production control of darobactins, due to their autotoxicity against the Gram-negative host. While there was no significant difference in D22 production titre when comparing E. coli BL21 (DE3) (10.5 (±1.0) mg/L) to E. coli BL21-Gold (DE3) (9.0 (±1.2) mg/L), we opted to use E. coli BL21-Gold (DE3). This allowed for a more controlled fermentation, and thus better reproducibility in contrast to significantly increased leaky expression in uninduced E. coli BL21 (DE3), as shown in Groß et al. An optimised purification procedure utilised weak cationexchange resin Dowex MAC-3 and ammonia elution instead of XAD16-N as previously published. With this modified workflow, following an initial C18 flash chromatography step, we were able to reduce the purification effort of the darobactins to a single preparative C18 step that resulted in an increase in D22 yield from 0.8 mg/L culture to 3.0 to 3.7 mg/mL (3.8 to 4.6-fold). The detailed purification procedure can be found in the supplementary information.