An antitumor agent thiocoraline is a thiodepsipeptide marine product derived from two Micromonospora sp. strains that inhibits protein synthesis by binding of its key 3-hydroxyquinaldic acid (3HQA) chromophores to duplex DNA. There are at least two potential pathways via which the 3HQA moiety could be biosynthesized from L-Trp. By biochemical characterization and by preparation of knockouts of an adenylation–thiolation enzyme, TioK, and of two type II thioesterases, TioP and TioQ, found in the thiocoraline biosynthetic gene cluster, we gained valuable insight into the pathway followed for the production of 3HQA.