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.
Highlights
To determine whether this cluster was involved in the production of astellolides, we disrupted each gene in a ΔcclA background
There are two types of terpene cyclases based on the production mechanism of the initial carbocation: (1) an “ionisation-initiated’’ mechanism, which generates a carbocation by the release of a pyrophosphate group via the conserved DDxxD/E motif; and (2) a “protonation-initiated” mechanism, which generates a carbocation by protonating a double-bond via the conserved DxDD (DxDTT) motif
Kwon et al reported the cloning and characterisation of a plant drimenol cyclase containing a typical ionisation-initiated motif[23]; the underlying catalytic mechanism was not elucidated in their study
Summary
To examine whether AstC had sesquiterpene cyclase activity, we purified it (Fig. 2a) and performed the AstC reaction in the presence of farnesyl pyrophosphate (FPP). Incubation of AstI with the AstC reaction mixture resulted in a significant increase in the Pi concentration (Fig. 3d). To identify the missing drimanyl monophosphate dephosphorylase, we purified AstK (Fig. 3a) and incubated it together with AstI in the presence of the AstC reaction mixture.
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