A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the beta-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis.