Identification and Characterization of a Cryptic Bifunctional Type I Diterpene Synthase Involved in Talaronoid Biosynthesis from a Marine-Derived Fungus

Abstract

We report the identification of the tnd biosynthetic cluster from the marine-derived fungus Aspergillus flavipes and the in vivo characterization of a cryptic type I diterpene synthase. The heterologous expression of the bifunctional terpene synthase led to the discovery of a diterpene backbone, talarodiene, harboring a benzo[ a ]cyclopenta[ d ]cyclooctane tricyclic fused ring system. The conversion of geranylgeranyl diphosphate to talarodiene was investigated using 13 C-labeling studies, and stable isotope tracer experiments showed the biotransformation of talarodiene into talaronoid C.

Biosynthesis of fungal terpenoids

“…avipes CNL-338 is the only example that proceeds through a type-c cyclization cascade, which forms the 5/8/6 tricyclic diterpene talarodiene (54). 81 The cyclization from G + involves ring expansion to form G-8 + . Subsequent C10 deprotonation and reprotonation at C2 generate G-9 + , which then undergoes 1,2-hydride transfer and C2-C6 cyclization to form G-10 + .…”

“…Deprotonation of G-10 + at C8 yields 54 (Scheme 8). 81 2.1.3.3 Diterpene cyclases with type-d cyclization mechanism. The cyclization cascades of the terpenoid cyclases with typed cyclization mechanism from cation G + proceed through two successive WMR via ring expansion and contraction to form cation G-11 + .…”

Conformational Control in Diterpene Synthase TlnA: Elucidating the Mechanism of 5–8–6 Skeleton Formation through Ring Expansion

“…3 The crystal structure and cyclization mechanism of PaFS have been extensively studied, offering deep insights into the enzymatic facilitation of 5−8−5 skeleton formation. 4 Building upon the foundational understanding on PaFS, our previous research introduced TndC, 5 the first diterpene synthase identified to facilitate the formation of the diterpene talarodiene with a 5−8−6 skeleton, marking a significant stride in the field. However, the in vitro functionality and detailed cyclization mechanism of TndC remain largely unexplored, opening a vista of opportunities for further research.…”

“…Upon normal-phase high-performance liquid chromatography (HPLC), two new products ( 1 and 2 ) were observed in the metabolites from AO- tlnA (Figure B). The major product ( 1 ) was identified as talarodiene, and the minor product ( 2 ) was confirmed as a new product; their structures were determined on the basis of the nuclear magnetic resonance (NMR) data (Figures S13–S20). Then, we prepared a recombinant TlnA protein in Escherichia coli for an in vitro enzymatic reaction.…”

Hot off the press

“…The gene cluster responsible for the biosynthesis of talaronoid C 28 has been characterised from the marine-derived fungus Aspergillus flavipes . 24 Heterologous expression of cryptic bifunctional type I diterpene synthase from this pathway identified talarodiene 29 as an intermediate of talaronoid C biosynthesis.…”