Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein–protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an α-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or α-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.