Isolation of a gene cluster from Armillaria gallica for the synthesis of armillyl orsellinate–type sesquiterpenoids

Abstract

Melleolides and armillyl orsellinates are protoilludene-type aryl esters that are synthesized exclusively by parasitic fungi of the globally distributed genus Armillaria (Agaricomycetes, Physalacriaceae). Several of these compounds show potent antimicrobial and cytotoxic activities, making them promising leads for the development of new antibiotics or drugs for the treatment of cancer. We recently cloned and characterized the Armillaria gallica gene Pro1 encoding protoilludene synthase, a sesquiterpene cyclase catalyzing the pathway-committing step to all protoilludene-type aryl esters. Fungal enzymes representing secondary metabolic pathways are sometimes encoded by gene clusters, so we hypothesized that the missing steps in the pathway to melleolides and armillyl orsellinates might be identified by cloning the genes surrounding Pro1. Here we report the isolation of an A. gallica gene cluster encoding protoilludene synthase and four cytochrome P450 monooxygenases. Heterologous expression and functional analysis resulted in the identification of protoilludene-8α-hydroxylase, which catalyzes the first committed step in the armillyl orsellinate pathway. This confirms that ∆-6-protoilludene is a precursor for the synthesis of both melleolides and armillyl orsellinates, but the two pathways already branch at the level of the first oxygenation step. Our results provide insight into the synthesis of these valuable natural products and pave the way for their production by metabolic engineering. Key points • Protoilludene-type aryl esters are bioactive metabolites produced by Armillaria spp. • The pathway-committing step to these compounds is catalyzed by protoilludene synthase. • We characterized CYP-type enzymes in the cluster and identified novel intermediates.

Deep Learning Approach for Predicting the Therapeutic Usages of Unani Formulas towards Finding Essential Compounds

“…Gibberellin A12 is implicated in the treatment of male infertility [ 33 ]. The ∆-6-protoilludene is a precursor for the synthesis of both melleolides and armillyl orsellinates, whose cytotoxicity reflects their ability to induce apoptosis [ 34 ]. In addition, erythrodiol is an active ingredient from the herb, Rhododendron ferrugineum , which is used in TCM.…”

A Combination of Genome Mining with an OSMAC Approach Facilitates the Discovery of and Contributions to the Biosynthesis of Melleolides from the Basidiomycete Armillaria tabescens

“…Nevertheless, the pioneering biogenesis insights were only preliminary. Until now, very few studies were reported for investigating the biosynthetic process of such structurally unique molecules. ,, Lackner and coworkers reconstituted the biosynthesis of melledonol and demonstrated that the ArmB could accept varied alcohols as substrates . Recently, Ichinose et al reported three cytochrome P450 monooxygenases CYP5344B1, CYP5348E1, and CYP5348J3 from the basidiomycete Postia placenta ATCC 44394, which facilitated the hydroxylation of Δ6-protoilludene to generate Δ6-protoilludene-3-ol or Δ6-protoilludene-5-ol, respectively.…”

“…This genus has been widely applied as nutraceutical medicinal mushrooms and is considered to be a treasure for melleolide discovery. , Since the report of the first orsellinic acid-derived melleolide isolated from A. mellea decades ago, a large number of bioactive protoilludane sesquiterpene aryl esters have been characterized, and the compounds of this family have recently gained interest. ,, Armillaria is a distinctive fungal genus due to its metabolic versatility and capability to decompose a diversity of plant cell wall components as recently highlighted by Sipos et al Genome mining of the strain CPCC 401429 revealed the presence of a melleolide biosynthetic gene cluster. It carries the UDPGT coding gene melO , which is hypothesized to be of producing glycosylated molecules.…”

“…The ensemble concludes genes that encode a terpene synthase (TS) and a NR-PKS 14 . The prevalence of these PTase-containing biosynthetic clusters among these agaricales gives reason to hypothesize that they might produce a group of structurally related melleolides of common biosynthetic origin. , Conspicuously, the mel locus is the shortest among these seven melleolide gene clusters and a delicate biosynthetic cluster (∼59 kb), the genes of which are clustered tightly (Figure ). In contrast, six other gene clusters are much larger (>81 kb), and encoding ORFs are scattered and distributed separately (Figure ).…”

“…54 In addition, CYP-Arm3 was characterized from A. gallica FU02472, which catalyzed in C-3 hydroxylation of Δ6protoilludene. 55 A phylogenetic dendrogram of four cytochrome P450s recovered based on gene cluster mel and characterized P450s was constructed, exhibiting a clear separation of fungal P450s (Figure 5). These P450s may contribute to the hydroxylation or consecutive hydroxylations of benzene rings, cycloalkanes, and cycloolefines.…”