BE-14106 and aureoverticillactam represent small-ring macrocyclic lactams with an acyl side chain, which possess anti-tumor activity. We have isolated Streptomyces spp. from marine sediments producing BE-14106 and its close homologue ML-449 with extended acyl side chain. The respective gene clusters have been cloned, sequenced and analyzed using bioinformatics, gene inactivation experiments, and heterologous expression of certain genes followed by enzymatic assays. The most interesting feature of the biosynthesis of these macrolactams appears to be the process of the aminoacyl starter biosynthesis, which involves the concerted action of a distinct polyketide synthase (PKS) system and enzymes for activation and oxidative deamination of an amino acid. Comparison of the BE-14106 and ML-449 biosynthetic pathways indicated that the difference in the compounds’ structures stems from the incorporation of one extra acetate unit during the synthesis of the aminoacyl starter. A phylogenetic analysis of the ketosynthase domains from the PKS involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Comparison of homologous genes and the organization 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 macrolactam biosynthesis.