Identification of a novel sesquiterpene biosynthetic machinery involved in astellolide biosynthesis

Abstract

Esterified drimane-type sesquiterpene lactones such as astellolides display various biological activities and are widely produced by plants and fungi. Given their low homology to known sesquiterpene cyclases, the genes responsible for their biosynthesis have not been uncovered yet. Here, we identified the astellolide gene cluster from Aspergillus oryzae and discovered a novel sesquiterpene biosynthetic machinery consisting of AstC, AstI, and AstK. All these enzymes are annotated as haloacid dehalogenase-like hydrolases, whereas AstC also contains a DxDTT motif conserved in class II diterpene cyclases. Based on enzyme reaction analyses, we found that AstC catalysed the protonation-initiated cyclisation of farnesyl pyrophosphate into drimanyl pyrophosphate. This was successively dephosphorylated by AstI and AstK to produce drim-8-ene-11-ol. Moreover, we also identified and characterised a unique non-ribosomal peptide synthetase, AstA, responsible for esterifying aryl acids to drimane-type sesquiterpene lactones. In this study, we highlight a new biosynthetic route for producing sesquiterpene and its esterified derivative. Our findings shed light on the identification of novel sesquiterpenes via genome mining.

Taxonomy ofAspergillussectionFlaviand their production of aflatoxins, ochratoxins and other mycotoxins

“…1990 ), aspergillomarasmins ( Robert et al., 1962 , Barbier et al., 1963 ), asperopterin A & B ( Matsuura et al . 1972 ), aspirochlorins ( Sakata et al., 1983 , Champhamjon et al., 2014 ), cyclopiazonic acid and speradines ( Orth, 1977 , Tokuoka et al., 2015 ), 14-deacetyl parasiticolide A & B and dideacetyl parasiticolide A, confertifolin, dideacetyl astellolide A & B ( Rank et al., 2012 , Shinohara et al., 2016a , Shinohara et al., 2016b ), 13-desoxypaxilline ( Rank et al . 2012 ), ditryptoleucine ( Rank et al .…”

Nanangenines: drimane sesquiterpenoids as the dominant metabolite cohort of a novel Australian fungus, Aspergillus nanangensis

“…Therefore, the drimane synthase is likely to be different from the commonly observed sesquiterpene synthase, which belongs to the class I terpene synthases. Recently, the drimane synthase AstC involved in biosynthesis of the astellolides was identified and shown to be a novel member of the terpene synthase family, showing similarity to haloacid dehalogenase (HAD)-like hydrolases [21]. Thus, we suspected that a related enzyme may be involved in the biosynthesis of the nanangenines, and used the amino acid sequence of AstC to probe the A. nanangensis genome.…”

“…AstC, though annotated as a HAD-like hydrolase and having low sequence homology to other characterised terpene synthases, contains sequence motifs conserved across both class I (DDxxD/E) and class II (DxDD, QW) terpene synthases [33–35]. Specifically, AstC contains a DxDTT motif [21], which is a variant of the DxDD motif known to be involved in class II-type protonation-initiated terpene cyclisation [36]. Importantly, the DDxxD motif in AstC contains a substitution of the second Asp for Asn, leading to a loss of ionisation-activated cyclisation activity.…”

“…The fungal genus Aspergillus is well recognised as a source of structurally diverse terpenoids comprising monoterpenoids [1], sesquiterpenoids [2–5], diterpenoids [6], sesterterpenoids [7–9], triterpenoids [10] and prenylated polyketide meroterpenoids [11–15] isolated from soil, endophytes and marine strains. Of this genus, A. ustus [16], A. calidoustus [17], A. insuetus [17], A. insulicola [18], A. bridgeri [18], A. sclerotiorum [19], A. variecolor [19], A. parasiticus [20], A. oryzae [21], A. ochraceus [22], A. pseudodeflectus [17], A. carneus [23] and Aspergillus sp. strain IBWF002-96 [4–5] are biosynthetic sources of the drimane sesquiterpenoids.…”

Combined Comparative Genomics and Gene Expression Analyses Provide Insights into the Terpene Synthases Inventory in Trichoderma

“…These HAD-like proteins harbor a DxDTT motif, which is a variant of the DxDD found in Class II diTS [ 95 , 96 ]. Shinohara et al [ 97 ] reported that some sesquiTS can contain HAD-like domains and DxDTT motifs, leading to FPP cyclization through a protonation step, instead of by an ionization step. We hypothesized that HAD-like TSs might be particular bifunctional sesquiTS, synthesizing specific metabolites of Viride clade.…”