Biosynthetic origin of anticancer dolastatin 15 reveals a noncanonical NRPS architecture enabling 2-keto acid activation.

Elucidating the biological origins and biosynthetic pathways of marine natural products remains a major challenge, particularly for compounds derived from microbial consortia. Here, we identify the marine cyanobacterial genus Dapis as the source of the potent anticancer natural product dolastatin 15 using genome-resolved metagenomics. Reconstruction of two complete and two near-complete genomes provides genome-resolved characterization of the genus Dapis, revealing extensive biosynthetic potential. Genome mining identified the dolastatin 15 biosynthetic gene cluster. Subsequent analyses reconstructed its pathway and uncovered a noncanonical five-domain nonribosomal peptide synthetase module containing an embedded 2-keto acid-activating domain, revealing an unexpected architectural solution for incorporating 2-keto acid-derived hydroxy acid building blocks. Comparative and evolutionary analyses support a model in which this architecture may have arisen through recruitment of keto acid-activating domains and reduction of canonical adenylation domain features. We further biochemically and structurally characterize an O-methyltransferase that catalyzes formation of the characteristic methoxy pyrrolinone moiety, thereby validating a key terminal tailoring step in the proposed biosynthetic pathway. Together, these findings define the biosynthetic logic of dolastatin 15 and expand understanding of substrate activation and assembly-line diversification in nonribosomal peptide biosynthesis.