Exploring diterpene metabolism in non‐model species: transcriptome‐enabled discovery and functional characterization of labda‐7,13E‐dienyl diphosphate synthase from Grindelia robusta

Abstract

SUMMARYGrindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases

“…This secondary product is likely resulting from thermal degradation of copalol during GC-MS analysis as previously observed in transient expression analyses of other class II diTPSs [21, 24, 37, 46]. In contrast to IrTPS5, sequential activity of IrTPS3 was only detected with MvELS yielding miltiradiene (product e), whereas no product formation was detected after co-expression with GrEKS (Fig 3B).…”

“…Due to the natural pairwise activity of monofunctional class II and class I diTPSs in angiosperm labdane biosynthesis [16, 20, 34, 35], combinatorial expression of class II and class I diTPSs can be used to determine the identity and stereochemistry of enzyme products [21, 23, 24, 36–45]. To this end, we co-expressed IrTPS5 and IrTPS3 with previously characterized class I diTPSs that exhibited substrate-specificity to ent -CPP ( Grindelia robusta ent -kaurene synthase, GrEKS [37]) and (+)-CPP ( Marrubium vulgare epoxy-labdane/miltiradiene synthase, MvELS [21]), respectively. Transient expression of IrTPS5 alone yielded copalol (product a) as verified by comparison to an enzyme-produced standard (Fig 3A).…”

“…Production of copalol representing the dephosphorylated form of CPP is commonly observed in transient N . benthamiana co-expression assays of class II diTPSs and is presumably caused by endogenous phosphatases that dephosphorylate the class II diTPS products [17, 18, 21, 36, 37]. Subsequent co-expression of IrTPS5 with GrEKS afforded ent -kaurene (product b) as the sole product, while no product formation was observed after co-expression with MvELS.…”

“…All NMR spectra were acquired with TopSpin 3.2 software (Bruker, Billerica, MA) and analyzed with MestReNova 11.0.2 software (Mestrelab Research, Santiago de Compostela, Spain). Chemical shifts (S1 Fig) were calibrated against known chloroform ( 1 H 7.26 and 13 C 77.0 ppm) signals, and compared to previously reported chemical shifts for nezukol [35,37,38]. …”

A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum

“…These results not only confirm the function of Fl-SdKPS as KPS, but also indicate that (–)-kolavenyl diphosphate can be dephosphorylated to (–)-kolavenol by an endogenous enzyme from N. benthamiana leaves. It has been previously suggested that endogenous phosphatases can convert the product of class II diTPSs into corresponding diterpenols in tobacco (Sallaud et al , 2012; Zerbe et al , 2014, 2015). Thus, in vivo functional characterization of Fl-SdKPS confirmed the results obtained in vitro with Tr-SdKPS.…”