The biocontrol agent Lysobacter enzymogenes produces polycyclic tetramate macrolactams (PoTeMs), including the antifungal HSAF. To elucidate the biosynthesis of the cyclic systems, we identified eleven HSAF precursors/analogues with zero, one, two, or three rings through heterologous expression of the HSAF gene cluster. A series of combinatorial gene expression and deletion experiments showed that OX3 is the "gatekeeper" responsible for the formation of the first 5-membered ring from lysobacterene A, OX1 and OX2 are responsible for formation of the second ring but with different selectivity, and OX4 is responsible for formation of the 6-membered ring. In vitro experiments showed that OX4 is an NADPH-dependent enzyme that catalyzes the reductive cyclization of 3-dehydroxy alteramide C to form 3-dehydroxy HSAF. Thus, the multiplicity of OX genes is the basis for the structural diversity of the HSAF family, which is the only characterized PoTeM cluster that involves four redox enzymes in the formation of the cyclic system.

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 CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5–6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.

Polycyclic tetramate macrolactams (PoTeMs) comprise a family of pharmacologically promising macrolactams that possess a tetramic acid moiety and various carbocyclic ring systems. Despite the progress in elucidating the biosynthetic pathways of representative PoTeMs, the mechanism by which the bicyclic system is formed remains unclear. Here, we report the targeted discovery of 5−5 bicyclic pseudoamides A−C (3−5) through heterologous expression of the recombinant pel cluster, which is a novel PoTeM cluster for products bearing a C13−C20 cyclization pattern. We also demonstrated that two FMN-dependent oxidoreductases, Pel1 and Pel3, are required for the generation of the 5−5 bicyclic system. Pel1 catalyzes a reductive cyclization reaction to form the outer 5-membered ring, which involves the incorporation of the hydride of FMNH 2 and one proton from water. Pel3 represents the unique cyclase catalyzing a C13−C20 cyclization coupled with the C-12 hydroxylation and is proposed to transfer oxygen from FMN-C4a-hydroxyperoxide to the substrate. Thus, this work establishes key biochemical insights into the formation of the 5−5 bicyclic system in PoTeM biosynthesis.

The biocontrol agent Lysobacter enzymogenes produces polycyclic tetramate macrolactams (PoTeMs), including the antifungal HSAF.T oe lucidate the biosynthesis of the cyclic systems,w ei dentified eleven HSAF precursors/ analogues with zero,o ne,t wo,o rt hree rings through heterologous expression of the HSAF gene cluster.Aseries of combinatorial gene expression and deletion experiments showed that OX3i st he "gatekeeper" responsible for the formation of the first 5-membered ring from lysobacterene A, OX1and OX2are responsible for formation of the second ring but with different selectivity,a nd OX4i sr esponsible for formation of the 6-membered ring. In vitro experiments showed that OX4i sa nN ADPH-dependent enzyme that catalyzest he reductive cyclization of 3-dehydroxy alteramide Ctoform 3-dehydroxy HSAF.Thus,the multiplicity of OX genes is the basis for the structural diversity of the HSAF family,w hich is the only characterized PoTeMc luster that involves four redox enzymes in the formation of the cyclic system.

焦玉杰 †,a 颜雅倩 †,a 刘 焱 a 朱德裕 b 沈月毛 * ,a 李瑶瑶 * ,a ( a 山东大学药学院 天然产物化学生物学教育部重点实验室 济南 250012) ( b 山东大学基础医学院 济南 250012) 摘要 从链霉菌 S001 的重组菌株 S001-PoTeM S023 中分离获得 1 个结构新颖的含有 5/5/6 三环体系的多环特特拉姆酸大 环内酰胺(PoTeMs)类化合物 montamide A (1), 通过一维和二维核磁(NMR)、高分辨质谱和圆二色谱确定了其化学结构.