Molecular analysis of the aureothin biosynthesis gene cluster from streptomyces thioluteus HKI-227 : new insights into polyketide assemply
Aureothin is a rare nitroaryl-substituted metabolite from the soil bacterium Streptomyces thioluteus, which exhibits a variety of biological activities, including antifungal, antitumoral and insecticidal. By construction and screening of a S. thioluteus HKI-227 genomic cosmid library, the aureothin (aur) gene cluster was cloned and sequenced. Detailed sequence analyses revealed that aureothin is synthesized by an unusual modular type I polyketide synthase (PKS), which has several exceptional features, including a novel priming mechanism and the iterative use of one module. Typically, modular type I PKS are arranged in an assembly line fashion, harboring a catalytic domain for each biosynthetic step. In stark contrast to this accepted principle of co-linearity, heterologous expression of the aur PKS cluster and fusion of the first two PKS genes demonstrate that one module catalyzes two successive cycles of chain extension, one of the first examples of a PKS in which such iteration or "stuttering" is required to produce the normal polyketide product. Further functional analysis of the aur biosynthesis gene cluster revealed several additional ORFs encoding starter unit biosynthesis enzymes, most remarkably an unprecedented N-oxygenase, which represents the first member of a new family of oxidizing enzymes. Furthermore, the order of the tailoring reactions was elucidated by mutational biosynthesis.