Expression of the Clifednamide Biosynthetic Pathway in Streptomyces Generates 27,28- seco-Derivatives

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP Natural Products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the Cytochrome P450 enzyme Cft_S023A and the hydroxylase Cft_S023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.