SUMMARYTripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. The absence of activity-forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii.

Background: Eremophila R.Br. (Scrophulariaceae) is a diverse genus of plants with species distributed across semi-arid and arid Australia. It is an ecologically important genus that also holds cultural significance for many Indigenous Australians who traditionally use several species as sources of medicines. Structurally unusual diterpenoids, particularly serrulatane and viscidane-types, feature prominently in the chemical profile of many species and recent studies indicate that these compounds are responsible for much of the reported bioactivity. We have investigated the biosynthesis of diterpenoids in three species: Eremophila lucida, Eremophila drummondii and Eremophila denticulata subsp. trisulcata. Results: In all studied species diterpenoids were localised to the leaf surface and associated with the occurrence of glandular trichomes. Trichome-enriched transcriptome databases were generated and mined for candidate terpene synthases (TPS). Four TPSs with diterpene biosynthesis activity were identified: ElTPS31 and ElTPS3 from E. lucida were found to produce (3Z,7Z,11Z)-cembratrien-15-ol and 5-hydroxyviscidane, respectively, and EdTPS22 and EdtTPS4, from E. drummondii and E. denticulata subsp. trisulcata, respectively, were found to produce 8,9-dihydroserrulat-14-ene which readily aromatized to serrulat-14-ene. In all cases, the identified TPSs used the cisoid substrate, nerylneryl diphosphate (NNPP), to form the observed products. Subsequently, cis-prenyl transferases (CPTs) capable of making NNPP were identified in each species. Conclusions: We have elucidated two biosynthetic steps towards three of the major diterpene backbones found in this genus. Serrulatane and viscidane-type diterpenoids are promising candidates for new drug leads. The identification of an enzymatic route to their synthesis opens up the possibility of biotechnological production, making accessible a ready source of scaffolds for further modification and bioactivity testing.

Summary Vitex agnus‐castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus‐castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus‐castus. With the assistance of matrix‐assisted laser desorption ionisation‐mass spectrometry imaging (MALDI‐MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome‐specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus‐castus. The identified CYP, VacCYP76BK1, was found to catalyse 16‐hydroxylation of the diol‐diterpene, peregrinol, to labd‐13Z‐ene‐9,15,16‐triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan‐ and lactone‐containing diterpenoids that are present in this species.

Background: Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. Results:To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. Conclusion:Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.

The medicinal plant Tripterygium wilfordii (Celastraceae) contains a pair of class II diterpene synthases (diTPS) of specialized labdane-type metabolism that, despite remarkably close homology, form strikingly different products. TwTPS21 catalyzes bicyclization of the linear C20 precursor geranylgeranyl diphosphate to ent-copal-8-ol diphosphate, while TwTPS14 forms kolavenyl diphosphate. To determine the amino acid signature controlling the functional divergence of the homologues, we modeled their structures based on an existing crystal structure of the Arabidopsis ent-copalyl diphosphate synthase, archetypal of diTPSs in general metabolism of gibberellin phytohormones. Of the residues differing between TwTPS21 and TwTPS14 two located to the predicted active site, and we hypothesized that these are responsible for the functional differentiation of the enzymes. Using site-directed mutagenesis, we generated a panel of six variants, where one, or both positions were exchanged between the enzymes. In coupled heterologous assays with a corresponding class I diTPS, TwTPS2, complete product interchange was observed in variants with both reciprocal mutations, while substitutions of either residue gave mixed product profiles. Two mutants, TwTPS14:Y265H and TwTPS21:A325V, also produced ent-copalyl diphosphate, highlighting the evolutionary potential of enzymes of this family to drive rapid diversification of plant diterpene biosynthesis through neo-functionalization. Our study contributes to the understanding of structure-function relation in plant class II diTPSs and complements previous mutational studies of Arabidopsis ent-copalyl diphosphate synthase with additional examples from the specialized metabolism of T. wilfordii.