An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping

Abstract

Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

A Family of Related Fungal and Bacterial Di‐ and Sesterterpenes: Studies on Fusaterpenol and Variediene

“…This finding suggests that the whole clade of enzymes shaded in grey in Figure might have activity as variediene synthases. The absolute configuration was investigated by using the enantioselective‐deuteration approach and independently by optical rotary power measurement; both pointed to the same configuration as published for 3 from E. variecolor (Figures S19 and S20) . The stereochemical fate of the geminal methyl groups C16 and C17 was in agreement with the observations made for the previously investigated diterpene synthases (Scheme , Figure S21).…”

“…All these compounds have a cis ‐fused cyclopentane ring that arises biosynthetically from geranylgeranyl diphosphate (GGPP) or geranylfarnesyl diphosphate (GFPP) by an initial 1,11–10,14 cyclisation to the cationic intermediate A and a subsequent ring expansion with simultaneous ring contraction to the hypothetical secondary cation B that can stabilise by further 2,10 or 6,10 cyclisation (Scheme A). The known fungal representatives are generally made by bifunctional enzymes containing a terpene synthase domain and a prenyltransferase domain for GGPP or GFPP biosynthesis (TS+PT) and include phomopsene ( 1 ) and its derivative methyl phomopsenoate ( 2 ) from Phomopsis amygdali , variediene ( 3 ) from Emericella variecolor and the cyclopiane‐type diterpene ( 4 ) from Penicillium chrysogenum (Scheme B) . Compound 4 is the biosynthetic precursor of a group of oxidised derivatives such as conidiogenone ( 5 ), one of the first isolated compounds of this class that exhibits an interesting bioactivity as a potent inducer of conidiogenesis in Penicillium …”

“…The absolute configurations of 2 , 3 , 7 and 12 were determined by modified Mosher's method, and the absolute configuration of fungal 1 was deduced from that of 2 . The absolute configurations of bacterial 1 and the other bacterial diterpene synthase products 13 – 15 were determined by chemical correlation using enantioselectively deuterated GGPP isotopomers .…”

“…To further investigate this point for fungal enzymes, the gene of a bifunctional TS+PT enzyme from A. brasiliensis CBS 101.740 (accession no. OJJ72250) with the published variediene synthase EvVS as closest characterised homologue (Figure , 65 % identical residues) was cloned and expressed in E. coli (Table S3, Figures S9 and S10). The purified enzyme converted a combination of DMAPP and IPP, and also GGPP, into 3 as a single product whose structure was resolved by NMR spectroscopy (Table S4 and Figures S11–S18), thus identifying this enzyme as A. brasiliensis variediene synthase (AbVS).…”

“…Previously characterised enzymes are shown in green, the enzyme characterised in this study is shown in red. AcOS: Aspergillus clavatus ophiobolin F synthase, CgDS: Colletotrichum gloeosporioides dolasta‐1(15),8‐diene synthase, EvAS: E. variecolor astellifadiene synthase, EvQS: E. variecolor quiannulatene synthase, EvSS: E. variecolor stellata‐2,6,19‐triene synthase, EvVS: E. variecolor variediene synthase, FgMS: F. graminearum mangicdiene synthase, NfSS: Neosartorya fischeri sesterfisherol synthase, PaFS: P. amygdali fusicocca‐2,10(14)‐diene synthase, PaPS: P. amygdali phomopsene synthase, PbSS: Penicillium brasilianum sesterbrasiliatriene synthase, PcCS: P. chrysogenum cyclopiane‐type diterpene synthase, PvPS: Penicillium verruculosum preasperterpenoid A synthase …”