The opportunistic pathogen Burkholderia thailandensis produces a number of structurally similar unsaturated quinolones involved in quorum sensing. However, little is known about the biosynthesis of these unsaturated quinolones. In this study, we have characterized the starting point of the biosynthesis of unsaturated quinolone molecules produced in B. thailandensis. We have shown by using in vitro enzymology, liquid chromatography, and mass spectrometry that protein HmqF is involved in the biosynthesis of unsaturated quinolones produced by B. thailandensis. HmqF consists of three domains: an adenylation domain (A domain), a dehydrogenase domain (DH domain), and an acyl carrier domain (ACP). The three domains (A, DH, and ACP) were cloned and expressed individually in Escherichia coli, and their reactivity was studied using high-performance liquid chromatography (HPLC) and mass spectrometry (MS) based assays. Our in vitro studies show that the A domain catalyzes ATP-dependent activation of medium chain (C6-C14) fatty acids without activation by coenzyme A (CoA). Results from competition assays are consistent with decanoic acid being the preferred substrate. Incubation of the ACP domain with 4'-phosphopantetheine transferase and CoA led to the formation of phosphopantetheinylated ACP (Ppant-ACP). In a Ppant ejection assay using tandem MS (MS/MS), a mass consistent with the mass of a cyclic variant of dephosphorylated Ppant was detected. We further demonstrated that Ppant-ACP could be loaded with medium chain fatty acids in the presence of ATP and the A domain. MS analysis was consistent with the formation of Ppant-ACP thiol esters of the fatty acids. MS/MS Ppant ejection experiments confirmed the loss of 2H in samples of fatty acid-loaded Ppant-ACP in the presence of the DH domain. HPLC analysis of benzyl amide ligation products allowed us to conclude that dehydrogenation produced trans-β,γ-unsaturation in the fatty acid chains. Our results are in good agreement with naturally observed quinolone molecules produced by B. thailandensis, which predominately produce nine-carbon trans-β,γ-unsaturated alkyl chain quinolone molecules.