Echosides, isolated from Streptomyces sp. LZ35, represent a class of para-terphenyl natural products that display DNA topoisomerase I and IIα inhibitory activities. By analyzing the genome draft of strain LZ35, the ech gene cluster was identified to be responsible for the biosynthesis of echosides, which was further confirmed by gene disruption and HPLC analysis. Meanwhile, the biosynthetic pathway for echosides was proposed. Furthermore, the echA-gene, encoding a tri-domain nonribosomal peptide synthetase (NRPS)-like enzyme, was identified as a polyporic acid synthetase and biochemically characterized in vitro. This is the first study to our knowledge on the biochemical characterization of an Actinobacteria quinone synthetase, which accepts phenylpyruvic acid as a native substrate. Therefore, our results may help investigate the function of other NRPS-like enzymes in Actinobacteria.