Releasing the potential power of terpene synthases by a robust precursor supply platform

Abstract

Terpenoids represent the largest family of natural products. Their structural diversity is largely due to variable skeletons generated by terpene synthases. However, terpene skeletons found in nature are much more than those generated from known terpene synthases. Most promiscuous terpene synthases (i.e. those that can generate more than one product) have not been comprehensively characterised. Here, we first demonstrated that the promiscuous terpene synthases can produce more variable terpenoids in vivo by converting precursor polyisoprenoid diphosphates of different lengths (C, C, C, C). To release the synthetic potential of these enzymes, we integrated the engineered MVA pathway, combinatorial biosynthesis, and point mutagenesis to depict the comprehensive product profiles. In total, eight new terpenoids were characterised by NMR and three new skeletons were revealed. This work highlights the key role of metabolic engineering for natural product discovery.

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

“…In contrast, a monofunctional diterpene synthase from F. graminearum gave a mixture of compounds with the diterpene alcohol 8 as main product, a compound that was provisionally designated by us GJ1012E and for which we now propose the name fusaterpenol. Side products made by the fusaterpenol synthase FgGS include 3 , its double‐bond isomer 9 and the related compounds 10 and 11 . Spirocyclic 11 is structurally similar to spirograterpene A ( 12 ) from Penicillium granulatum and to the bacterial compound spiroviolene ( 13 ) from Streptomyces violens , for which the diterpene synthase has recently been reported .…”

“…Recently, a bifunctional sesterterpene synthase for mangicdiene ( 6 ) was identified from Fusarium graminearum . This compound is the likely biosynthetic precursor for mangicol A ( 7 ) and other mangicols found in a marine Fusarium isolate .…”

“…Compound 3 from Fusarium was shown to have the same absolute configuration as 3 from E. variecolor based on a comparison of their optical rotations. Finally, the absolute configuration of 8 was obtained from crystal structure data, but was based on a fairly poor Flack parameter (−0.1(5)) . Notably, the stereochemistry at the initially formed cyclopentane ring of all these molecules is the same, only for compound 8 is a different stereochemistry found (grey shaded area in Scheme B); more importantly, this stereochemistry does not fit the configurations of the other products 3 and 9 – 11 of FgGS.…”

“…The proposed biosynthesis of 8 from GGPP through cyclisation to cation A , skeletal rearrangement to B , 2,10‐cyclisation to C , 3,7‐cyclisation and likely simultaneous attack of water to avoid a secondary cation intermediate was further studied by the enzymatic conversion of all 20 isotopomers of ( 13 C)GGPP (Table S2) with FgGS; the 13 C label was incorporated into the expected positions in all cases (Figure ). In particular, a clear stereochemical course for the geminal methyl groups C16 and C17 of GGPP was observed, with C16 ending up in the pro ‐ S and C17 in the pro ‐ R position, thus suggesting that the substrate is bound to the enzyme's active site with a precisely defined conformational fold.…”

“…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 …”