Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization

Abstract

Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum. A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.secondary metabolism | gene cluster | cytoxicity | emodin | nataloe-emodin

Monomeric Dihydroanthraquinones: A Chemoenzymatic Approach and its (Bio)synthetic Implications for Bisanthraquinones

“…Compound 3 is formed by the reduction of tautomers of emodin hydroquinone by NADPH‐dependent MdpC (from the monodictyphenone biosynthetic gene cluster of A. nidulans ), which on elimination of water results in the formation of chrysophanol ( 2 ). The same transformation is catalyzed by analogous enzymes, AflM from Aspergillus parasiticus and ‘17β‐hydroxysteroid dehydrogenase“ (17β‐HSDcl) from Cochliobolus lunatus , which further led to the elucidation of deoxygenation steps in aflatoxin B 1 and cladofulvin biosynthesis …”

“…The same transformationi sc atalyzed by analogous enzymes, AflM from Aspergillus parasiticus [11] and '17b-hydroxysteroid dehydrogenase" (17b-HSDcl) from Cochliobolus lunatus, [12] which further led to the elucidation of deoxygenation steps in aflatoxinB 1 [11] and cladofulvin biosynthesis. [13] Another related biosynthetic intermediate, dihydroemodin (4a), has been proposeda sb eing involved in the biosynthesis of (À)-flavoskyrin (5)a nd (À)-rugulosin (6), isolated from P. islandicum,a nd (+ +)-rugulosin (7), isolated from P. rugulosum. [14,15] The proposal was based on as eries of monomeric anthraquinones synthesized by Zahn and Koch almost 80 years ago, which tend to dimerize through ah etero-Diels-Alder reaction into flavoskyrin-type dimericc ompounds.…”

Down‐regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato

“…Tomato plants were inoculated with the wild‐type parental strain of C. fulvum , two independent Δ clag deletion mutants that are deficient in cladofulvin production only, and an ectopic insertion transformant control. These genetically modified strains have been obtained previously and have been characterized in vitro (Griffiths et al ., ). The resulting disease symptoms were similar for all strains, with each producing visible signs of conidiation from 12 dpi onwards (Figs a, a,b).…”

“…The wild‐type trace is reprinted from Griffiths et al . (), with permission from Elsevier. (c) Expression of the cladofulvin gene cluster during infection of tomato by the OE.claE transformant is compared with the in planta expression in the wild‐type.…”

Classic fungal natural products in the genomic age: the molecular legacy of Harold Raistrick

“…Chrysophanol 35 is also the substrate for hydroxylation, e.g. at C-7 to provide nataloe emodin 36, the precursor of cladofulvin 37, 41 or at C-4 to provide the isomeric islandicin 38 (Raistrick 1949). …”