Project description:Natural products with novel chemistry are urgently needed to battle the continued increase in microbial drug resistance. Mushroom-forming fungi are underutilized as a source of novel antibiotics in the literature due to their challenging culture preparation and genetic intractability. However, modern fungal molecular and synthetic biology tools have renewed interest in exploring mushroom fungi for novel therapeutic agents. The aims of this study were to investigate the secondary metabolites of nine basidiomycetes, screen their biological and chemical properties, and then investigate the genetic pathways associated with their production. Of the nine fungi selected, Hypholoma fasciculare was revealed to be a highly active antagonistic species, with antimicrobial activity against three different microorganisms: Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. Genomic comparisons and chromatographic studies were employed to characterize more than 15 biosynthetic gene clusters and resulted in the identification of 3,5-dichloromethoxy benzoic acid as a potential antibacterial compound. The biosynthetic gene cluster for this product is also predicted. This study reinforces the potential of mushroom-forming fungi as an underexplored reservoir of bioactive natural products. Access to genomic data, and chemical-based frameworks, will assist the development and application of novel molecules with applications in both the pharmaceutical and agrochemical industries.
Project description:Twelve compounds (1⁻12) were isolated from the methanol extract of brick cap mushroom (Hypholoma lateritium (Schaeff.) P. Kumm.). The structures of the compounds were elucidated using extensive spectroscopic analyses, including NMR and MS measurements. Lanosta-7,9(11)-diene-12β,21α-epoxy-2α,3β,24β,25-tetraol (1) and 8-hydroxy-13-oxo-9E,11E-octa-decadienoic acid (2) were identified as new natural products, together with ten known compounds, from which 3β-hydroxyergosta-7,22-diene (4), demethylincisterol A2 (5), cerevisterol (6), 3β-O-glucopyranosyl-5,8-epidioxyergosta-6,22-diene (7), fasciculol E (9), and uridine (12) were identified in this species for the first time. The isolated triterpenes (1, 3⁻11) were investigated for their toxicity in vivo using bdelloid rotifer assays. Most of the examined steroids in general showed low toxicity, although the effects of the compounds varied in a wider range from the non-toxic lanosta-7,9(11)-diene-12β,21α-epoxy-2α,3β,24β,25-tetraol (1) to the significantly toxic cerevisterol (6), with substantial dependence in some cases on the presence of nutrient in the experimental environment.
Project description:Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5β,6α,8β-trihydroxycariolan, 5β,8β-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.
