Enfumafungin synthase represents a novel lineage of fungal triterpene cyclases

Abstract

Summary Enfumafungin is a glycosylated fernene-type triterpenoid produced by the fungus Hormonema carpetanum. Its potent antifungal activity, mediated by its interaction with β−1,3-glucan synthase and the fungal cell wall, has led to its development into the semi-synthetic clinical candidate, ibrexafungerp (=SCY-078). We report on the preliminary identification of the enfumafungin biosynthetic gene cluster (BGC) based on genome sequencing, phylogenetic reconstruction, gene disruption, and cDNA sequencing studies. Enfumafungin synthase (efuA) consists of a terpene cyclase domain (TC) fused to a glycosyltransferase (GT) domain and thus represents a novel multifunctional enzyme. Moreover, the TC domain bears a phylogenetic relationship to bacterial squalene–hopene cyclases (SHC) and includes a typical DXDD motif within the active centre suggesting that efuA evolved from SHCs. Phylogenetic reconstruction of the GT domain indicated that this portion of the fusion gene originated from fungal sterol GTs. Eleven genes flanking efuA are putatively involved in the biosynthesis, regulation, transport and self-resistance of enfumafungin and include an acetyltransferase, three P450 monooxygenases, a dehydrogenase, a desaturase and a reductase. A hypothetical scheme for enfumafungin assembly is proposed in which the E-ring is oxidatively cleaved to yield the four-ring system of enfumafungin. EfuA represents the first member of a widespread lineage of fungal SHCs.

Ten decadal advances in fungal biology leading towards human well-being

“…Due to the medicinal relevance and unique structure of enfumafungin, the biosynthesis of the compound has been partially investigated (Kuhnert et al 2018 ). For this purpose, the genome of producer strain ATCC 74,360 has been sequenced with Illumina technology and assembled into 129 contigs with a total lengths of 32.8 Mbp.…”

Comparative Genomic and Metabolomic Analysis of Termitomyces Species Provides Insights into the Terpenome of the Fungal Cultivar and the Characteristic Odor of the Fungus Garden of Macrotermes natalensis Termites

“…Tri-TCs, which are key enzymes in ergosterol biosynthesis, carry a QW motif that determines its cyclization specificity in the transformation of squalene epoxide via a type II cyclization mechanism ( 9 , 10 ). So far only four Tri-TC candidates involved in the biosynthesis of triterpenoid natural products have been characterized from different fungal species ( 44 ).…”

Secondary metabolite biosynthetic diversity in the fungal family Hypoxylaceae and Xylaria hypoxylon

“…The first type corresponds to lanosterol synthases, which use oxidosqualene to form the tetracyclic lanosterol or structurally related backbones as found in helvolic acid and fusidic acid ( Hu et al. 2020 ), while fernane synthases use the same substrate to yield pentacyclic fernanes, the precursor of compounds such as enfumafungin or polytolypin ( Kuhnert et al. 2018 ).…”

“…Interestingly, the underlying BGC was distantly located from this LSS, demonstrating that localisation of fungal triterpene BGCs can be challenging. We also recently reported the ubiquitous presence of a new type of fungal triterpene synthases, the squalene-hopene cyclases (SHC), which have been linked to the production of the potent antifungal compound enfumafungin in Hormonema carpetanum ( Kuhnert et al. 2018 ).…”

“…It is therefore likely that H. rubiginosum is able to produce novel fernane glycosides under the correct conditions. This is in particular interesting as most of the known fernanes possess potent activities mainly against fungi and therefore could be of pharmaceutical relevance ( Kuhnert et al. 2018 ).…”