<jats:title>ABSTRACT</jats:title> <jats:p> Coronafacoyl phytotoxins are an important family of plant toxins that are produced by several different phytopathogenic bacteria, including the gammaproteobacterium <jats:named-content content-type="genus-species">Pseudomonas syringae</jats:named-content> and the actinobacterium <jats:named-content content-type="genus-species">Streptomyces scabiei</jats:named-content> (formerly <jats:named-content content-type="genus-species">Streptomyces scabies</jats:named-content> ). The phytotoxins consist of coronafacic acid (CFA) linked via an amide bond to different amino acids or amino acid derivatives. Previous work suggested that <jats:named-content content-type="genus-species">S. scabiei</jats:named-content> and <jats:named-content content-type="genus-species">P. syringae</jats:named-content> use distinct biosynthetic pathways for producing CFA, which is subsequently linked to its amino acid partner to form the complete phytotoxin. Here, we provide further evidence that the <jats:named-content content-type="genus-species">S. scabiei</jats:named-content> CFA biosynthetic pathway is novel by characterizing the role of CYP107AK1, a predicted cytochrome P450 that has no homologue in <jats:named-content content-type="genus-species">P. syringae</jats:named-content> . Deletion of the <jats:italic>CYP107AK1</jats:italic> gene abolished production of coronafacoyl-isoleucine (CFA-Ile), the primary coronafacoyl phytotoxin produced by <jats:named-content content-type="genus-species">S. scabiei</jats:named-content> . Structural elucidation of accumulated biosynthetic intermediates in the Δ <jats:italic>CYP107AK1</jats:italic> mutant indicated that CYP107AK1 is required for introducing the oxygen atom that ultimately forms the carbonyl group in the CFA backbone. The <jats:italic>CYP107AK1</jats:italic> gene along with two additional genes involved in CFA-Ile biosynthesis in <jats:named-content content-type="genus-species">S. scabiei</jats:named-content> were found to be associated with putative CFA biosynthetic genes in other actinobacteria but not in other organisms. Analysis of the overall genetic content and organization of known and putative CFA biosynthetic gene clusters, together with phylogenetic analysis of the core biosynthetic genes, indicates that horizontal gene transfer has played an important role in the dissemination of the gene cluster and that rearrangement, insertion, and/or deletion events have likely contributed to the divergent biosynthetic evolution of coronafacoyl phytotoxins in bacteria. <jats:p> <jats:bold>IMPORTANCE</jats:bold> The ability of plants to defend themselves against invading pathogens relies on complex signaling pathways that are controlled by key phytohormones such as jasmonic acid (JA). Some phytopathogenic bacteria have evolved the ability to manipulate JA signaling in order to overcome host defenses by producing coronatine (COR), which functions as a potent JA mimic. COR and COR-like molecules, collectively referred to as coronafacoyl phytotoxins, are produced by several different plant-pathogenic bacteria, and this study provides supporting evidence that different biosynthetic pathways are utilized by different bacteria for production of these phytotoxins. In addition, our study provides a greater understanding of how coronafacoyl phytotoxin biosynthesis may have evolved in phylogenetically distinct bacteria, and we demonstrate that production of these compounds may be more widespread than previously recognized and that their role for the producing organism may not be limited to host-pathogen interactions.