The Biogenetic Origin of the Biologically Active Naematolin of Hypholoma Species Involves an Unusual Sesquiterpene Synthase

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.

Further Biochemical Profiling of Hypholoma fasciculare Metabolome Reveals Its Chemogenetic Diversity

“…Seven verified constructs-pTYGS-argHfasTerp179, pTYG-argCop-1, pTYG-argCop-2, pTYG-argCop-3, pTYG-argCop-4, pTYG-argOmp6, and pTYG-argOmp7-were independently transferred to A. oryzae strain NSAR1 protoplasts via polyethylene glycol (PEG)-mediated transformation reactions. Subsequent gas chromatography-mass spectrometry (GC-MS) analysis on previous (pTYGS-argHfasTerp94a, pTYGS-argHfasTerp94b, and pTYGS-argHfasTerp344) and current transformants confirmed our previous results of HfasTerp94a, HfasTerp94b, and HfasTerp344 (Al-Salihi et al, 2019) through the replication of humulene and caryophyllene production, although at lower levels. The production of most of the major products of the two controls, pTYG-argCop-3 and pTYG-argCop-4, were confirmed as well as the previously uncharacterized humulene in the Cop-3 extract (Figure 8 and Supplementary Figure 33).…”

“…Our previous genomic investigation of Hypholoma suggested that only terpenoid compounds were produced, with a range of cyclization patterns ( Al-Salihi et al, 2019 ). However, a subsequent in-depth BLAST search of functionally characterized core enzymes selected from different fungi resulted in the identification of additional biosynthetic gene clusters (BGCs) in both Hypholoma species (see Supplementary Tables 1 , 2 ).…”

“…The previously described ethyl acetate metabolite extraction protocol was used ( Bailey et al, 2016 ). The chemical compositions of the wild type and the silenced lines (20 μl, final concentration of 5 mg/ml) of each crude extract were then compared by high-performance liquid chromatography (HPLC) as described in ( Al-Salihi et al, 2019 ).…”

“…The cDNA versions of the sesquiterpene synthases (Cop-1, Cop-2, Cop-3, Cop-4, Omph-6, and Omph-7) were kindly provided by Schmidt’s group ( Agger et al, 2009 ; Wawrzyn et al, 2012 ). Expression vectors were generated by yeast-based recombination as described in Al-Salihi et al (2019) . A. oryzae transformants were generated for the 10 selected enzymes and chemically analyzed using the protocol described in Al-Salihi et al (2019) .…”

“…work were used (Al-Salihi et al, 2019), as well as six previously characterized fungal sesquiterpene synthases from two different basidiomycetes, Omphalotus olearius and Coprinopsis cinerea, as controls: cop-1, cop-2, cop-3, cop-4, omp-6, and omp-7 (Agger et al, 2009;Wawrzyn et al, 2012). Seven verified constructs-pTYGS-argHfasTerp179, pTYG-argCop-1, pTYG-argCop-2, pTYG-argCop-3, pTYG-argCop-4, pTYG-argOmp6, and pTYG-argOmp7-were independently transferred to A. oryzae strain NSAR1 protoplasts via polyethylene glycol (PEG)-mediated transformation reactions.…”