Albicidin is a potent antibiotic and phytotoxin produced by the sugarcane pathogenic bacterium Xanthomonas albilineans which targets the plant and bacterial DNA gyrase.[1] The infection occurs through the xylem vessels of the plants where the pathogen induces chlorosis which is characterized by white-pencil streaks.[2] Albicidin is encoded in the alb gene cluster, a polyketide synthase non-ribosomal peptide synthetase hybrid (PKS-NRPS). In addition to the PKS-NRPS megasynthetase machinery their existing several tailoring and post-NRPS proteins, whose function in connection with the processing of albicidin is unclear.[3] We could characterize a new albicidin derivative with a combination of tandem mass spectrometry, in vitro and knockout experiments from cultures of a Xanthomonas overproducer which is carbamoylated at the N-terminal coumaric acid. The putative ATP-dependent O-carbamoyltransferase Alb15, present in the biosynthesis gene cluster of albicidin, is responsible for the derivatization. Cloning and Expression of Alb15 in Escherichia coli and in vitro reconstitution of the carbamoyltransferase activity confirmed albicidin as the target substrate. The chemical synthesis of carbamoyl-albicidin enabled us to carry out bioactivity assays by means of in vitro gyrase inhibition and antibacterial assays. Carbamoyl-albicidin showed a significantly higher inhibitory efficiency against bacterial gyrase (~8 nM vs 49 nM) then albicidin, which identifies the carbamoyl group as an important structural feature of albicidin maturation.