Abstract
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.
Highlights
With the evolving resistance to almost all existing antibiotics, there is an urgent need for new classes of antibiotics that are naturally produced [1].Lately, new classes of antibiotics have emerged based on terpene-based structures isolated from Basidiomycetes [2], but to date only a small number have been analysed in detail
We report the isolation of a candidate gene cluster for naematolin, including heterologous expression of the first two biosynthetic genes, paving the way for further combinatorial biosynthesis of naematolin-based isomers, potentially leading to the generation of new antibiotic classes
The antibacterial properties of naematolin were confirmed by disc diffusion assays, showing some limited activity against Bacillus subtilis and Staphylococcus aureus, but no activity against Pseudomonas aeruginosa or Klebsiella pneumoniae (Table S2)
Summary
With the evolving resistance to almost all existing antibiotics, there is an urgent need for new classes of antibiotics that are naturally produced [1]. New classes of antibiotics have emerged based on terpene-based structures isolated from Basidiomycetes [2], but to date only a small number have been analysed in detail. Naematolin is a bicyclic sesquiterpene thought to be derived from the caryophyllene scaffold of the 1,11 and 2,10 carbon cyclisation of farnesyl pyrophosphate. This metabolite was first reported in H. fasciculare by Ito and co-workers [3] where its chemical structure was established based on spectroscopic and limited NMR data [4, 5]. Biological investigations uncovered the antitumor and the antiviral properties of naematolin [6], but its production was in low titre and its complex structure precluded chemical synthesis, so the compound has remained comparatively unexploited
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