Abstract
Macrolactams comprise a family of natural compounds with important bioactivities, such as antibiotic, antifungal, and antiproliferative activities. Sipanmycins A and B are two novel members of this family, with two sugar moieties attached to the aglycon. In the related macrolactam vicenistatin, the sugar moiety has been proven to be essential for cytotoxicity. In this work, the gene cluster responsible for the biosynthesis of sipanmycins (sip cluster) in Streptomyces sp. strain CS149 is described and the steps involved in the glycosylation of the final compounds unraveled. Also, the cooperation of two different glycosyltransferases in each glycosylation step is demonstrated. Additionally, the essential role of SipO2 as an auxiliary protein in the incorporation of the second deoxy sugar is addressed. In light of the results obtained by the generation of mutant strains and in silico characterization of the sip cluster, a biosynthetic pathway for sipanmycins and the two deoxy sugars attached is proposed. Finally, the importance of the hydroxyl group at C-10 of the macrolactam ring and the sugar moieties for cytotoxicity and antibiotic activity of sipanmycins is shown.IMPORTANCE The rapid emergence of infectious diseases and multiresistant pathogens has increased the necessity for new bioactive compounds; thus, novel strategies have to be developed to find them. Actinomycetes isolated in symbiosis with insects have attracted attention in recent years as producers of metabolites with important bioactivities. Sipanmycins are glycosylated macrolactams produced by Streptomyces sp. CS149, isolated from leaf-cutting ants, and show potent cytotoxic activity. Here, we characterize the sip cluster and propose a biosynthetic pathway for sipanmycins. As far as we know, it is the first time that the cooperation between two different glycosyltransferases is demonstrated to be strictly necessary for the incorporation of the same sugar. Also, a third protein with homology to P450 monooxygenases, SipO2, is shown to be essential in the second glycosylation step, forming a complex with the glycosyltransferase pair SipS9-SipS14.