There is an urgent need for new antibiotics to maintain the high health standards to which we have got accustomed to because resistance against currently used antibiotics is increasing with an alarmingly high rate. It has recently been estimated that in 2019 alone there were 4.96 million deaths associated with antimicrobial resistance (AMR), including 1.27 million deaths attributable to bacterial AMR. To direct research efforts in this field, the World Health Organization has published a list of bacteria for which antibiotics are in high demand and urged that research and drug discovery efforts are directed toward these bacteria. Further, due to cross-resistance, ideally antibiotics acting on new targets are needed. A possible source for new targets for antibiotics is the fatty acid synthesis (FAS II) pathway (Figure ) which is highly conserved among bacterial pathogens, but distinct from the FAS I pathway in mammals. In several genetic screens, genes coding for enzymes in the FAS II pathway have been found to be essential. In addition, inhibition of the FAS II pathway has been shown to overcome resistance against the last resort antibiotic colistin. The drug isoniazid which targets the enoyl-ACP reductase FabI (Figure ) is in clinical use against tuberculosis, further validating this pathway. The FAS II pathway is also present in mitochondria of human cells, but its role is not entirely clear and short-term inhibition by antibiotics should be tolerated. Multiple enzymes in this pathway were predicted to contain druggable binding sites rendering them attractive targets for antibiotic drug discovery. Here, we focus on 3-oxoacyl-[acyl-carrier-protein] synthase 2 (also called beta-ketoacyl-ACP synthase 2 with FabF, Kas II, and KasB as commonly used short names). FabF catalyses the rate limiting Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP (Figure ). 3-oxoacyl-[acyl-carrierprotein] synthase 1 (FabB) catalyses the same reaction, but differs in substrate specificity. In the postulated reaction mechanism, the activated growing fatty acid chains first reacts with the negatively charged active site cysteine of FabF to form a covalent thioester intermediate. Subsequently, this intermediate reacts with malonyl-ACP to give a β-ketoacyl-ACP which is now elongated with two additional carbon atoms and at the same time to reconstitute the FabF enzyme. FabF has been validated in Gram-positive bacteria as the target for the natural products platensimycin, platencin and fasamycin A and B (Figure ). Platensimycin is inactive against wild-type Gram-negative bacteria, but active against efflux-negative E. coli. Similarly, the fasamycins are active against membrane-permeabilized E. coli but not the unaltered strain. These observations suggest that FabF is essential in Gram-negative bacteria, but that these natural products are not able to access the target due to permeability issues. Further target validation for FabF comes from the natural product inhibitors cerulenin and thiolactomycin (Figure ). Both natural products are effective against a range of bacteria and thiolactomycin was also efficacious in an animal model, however both lack selectivity and potency for clinical use. Platensimycin is the most potent FabF inhibitor known to date with activity in the low nanomolar range. . Most of its activity stems from binding to the fatty acid bound intermediate (Figure ) while binding to the w.t. enzyme is much weaker. The intermediate state can be mimicked by replacing the active side cysteine (Cys164 in PaFabF) with alanine or glutamine. Total syntheses of platencin and platensimycin have been carried out, and efforts have been undertaken to increase the antibiotic activity through synthesis of analogues. While some of these compounds showed promising activity against Gram-positive bacteria, for most compounds the potency activity against Gram-negative bacteria was not reported. In addition, poor pharmacokinetic properties were also a concern. Other potent ligands are therefore needed to further explore the potential of FabF as target in Gram-negative bacteria.