ABSTRACTMyxobacteria utilize the catechol natural products myxochelin A and B in order to maintain their iron homeostasis. Recently, the production of these siderophores, along with a new myxochelin derivative named pseudochelin A, was reported for the marine bacteriumPseudoalteromonas piscicidaS2040. The latter derivative features a characteristic imidazoline moiety, which was proposed to originate from an intramolecular condensation reaction of the β-aminoethyl amide group in myxochelin B. To identify the enzyme catalyzing this conversion, we compared the myxochelin regulons of two myxobacterial strains that produce solely myxochelin A and B with those ofP. piscicidaS2040. This approach revealed a gene exclusive to the myxochelin regulon inP. piscicidaS2040, coding for an enzyme of the amidohydrolase superfamily. To prove that this enzyme is indeed responsible for the postulated conversion, the reaction was reconstitutedin vitrousing a hexahistidine-tagged recombinant protein made inEscherichia coli, with myxochelin B as the substrate. To test the production of pseudochelin A underin vivoconditions, the amidohydrolase gene was cloned into the myxobacterial plasmid pZJY156 and placed under the control of a copper-inducible promoter. The resulting vector was introduced into the myxobacteriumMyxococcus xanthusDSM 16526, a native producer of myxochelin A and B. Following induction with copper, the myxobacterial expression strain was found to synthesize small quantities of pseudochelin A. Replacement of the copper-inducible promoter with the constitutivepilApromoter led to increased production levels inM. xanthus, which facilitated the isolation and subsequent structural verification of the heterologously produced compound.IMPORTANCEIn this study, an enzyme for imidazoline formation in pseudochelin biosynthesis was identified. Evidence for the involvement of this enzyme in the postulated reaction was obtained afterin vitroreconstitution. Furthermore, the function of this enzyme was demonstratedin vivoby transferring the corresponding gene into the bacteriumMyxococcus xanthus, which thereby became a producer of pseudochelin A. In addition to clarifying the molecular basis of imidazoline formation in siderophore biosynthesis, we describe the heterologous expression of a gene in a myxobacterium without chromosomal integration. Due to its metabolic proficiency,M. xanthusrepresents an interesting alternative to established host systems for the reconstitution and manipulation of biosynthetic pathways. Since the plasmid used in this study is easily adaptable for the expression of other enzymes as well, we expand the conventional expression strategy for myxobacteria, which is based on the integration of biosynthetic genes into the host genome.