(-)-Rugulosin

Herein, we report the chemoenzymatic synthesis of a heterodimeric (-)-rugulosin B, homodimeric (-)-rugulosin C, and several rugulin analogues in three to four steps starting from anthraquinones. This work supports dimerization between variously substituted putative monomeric intermediates during the biosynthesis of naturally occurring (+)-rugulosin B and C.

Disease-free second instar Choristoneura fumiferana (eastern spruce budworm) were placed on trees infected with a rugulosin-producing needle endophyte in two experiments. They were allowed to grow to sixth instar when survivors were collected. First, by using 3-yr old trees, a comparison was made of budworm growth on infected and uninfected trees. A second experiment used 4-yr old trees to study the effect of rugulosin content in the needle on growth. This permitted an examination of a dose response in relation to growth, and allowed us to eliminate the potential for differences in environment or foliar chemistry affecting the results. At sixth instar, budworms feeding on infected trees that contained rugulosin were smaller than those on uninfected trees. At needle concentrations above the dietary low observed effect level of rugulosin for C. fumerana >0.5 microg g(-1), a dose response was seen. For the first time, this demonstrates an inverse effect in outdoor nursery experiments between budworm weight and rugulosin concentration.

Skyrin and rugulosin A are bioactive bisanthraquinones found in many fungi, with the former suggested as a precursor of hypericin (a diversely bioactive phytochemical) and the latter characterized by its distinct cage-like structure. However, their biosynthetic pathways remain mysterious, although they have been characterized for over six decades. Here, we present theruggene cluster that governs simultaneously the biosynthesis of skyrin and rugulosin A inTalaromycessp. YE3016, a fungal endophyte residing inAconitum carmichaeli. A combination of genome sequencing, gene inactivation, heterologous expression, and biotransformation tests allowed the identification of the gene function, biosynthetic precursor, and enzymatic sets involved in their molecular architecture constructions. In particular, skyrin was demonstrated to form from the 5,5'-dimerization of emodin radicals catalyzed by RugG, a cytochrome P450 monooxygenase evidenced to be potentially applicable for the (chemo)enzymatic synthesis of dimeric polyphenols. The fungal aldo-keto reductase RugH was shown to be capable of hijacking the closest skyrin precursor (CSP) immediately after the emodin radical coupling, catalyzing the ketone reduction of CSP to inactivate its tautomerization into skyrin and thus allowing for the spontaneous intramolecular Michael addition to cyclize the ketone-reduced form of CSP into rugulosin A, a representative of diverse cage-structured bisanthraquinones. Collectively, the work updates our understanding of bisanthraquinone biosynthesis and paves the way for synthetic biology accesses to skyrin, rugulosin A, and their siblings.